Borisenko graduated from the Leningrad Physics and Mathematics School #30 in 1981. He then entered the Leningrad Military Mechanical Institute and graduated in 1987 with the qualification “Flight and Control Dynamics”.

Following graduation from the institute Borisenko worked for a military unit from 1987–1989. In 1989, he started working at RSC Energia where he was responsible for the Mir motion control system and took part in the Mission Control Center – Moscow (MCC-M) on board systems operation analysis board. In 1999, Borisenko was a shift flight director at the MCC-M, first for the Mir space station and then for the International Space Station (ISS).

Borisenko is currently scheduled to be one of the first Russian cosmonauts to fly on board an American Commercial Crew Vehicle, as he is the only confirmed member of a cadre of Russian cosmonauts assigned to fly on board either SpaceX’s Crew Dragon or Boeing’s Starliner spacecraft once they have completed crewed test flights and proved their safety. It is unclear which of the two spacecraft Borisenko will fly on as no cosmonauts have started training on either vehicle yet, and it has been stated by Pavel Vlasov (head of the Gagarin Cosmonaut Training Center) that if the two vehicles are further delayed it would be logical to revert Borisenko to a Soyuz flight. If Borisenko is the first Russian to fly on either Crew Dragon or Starliner, he will be the first Russian cosmonaut to launch on a spacecraft other than the Soyuz since Nikolai Budarin, who launched on the Space Shuttle on STS-113 in 2002.

Borisenko was selected as a cosmonaut candidate from RSC Energia on May 29, 2003. He started basic spaceflight training in June, 2003 and completed it in June, 2005 by passing the state exams with excellent grades. He received qualification of test-cosmonaut on July 5, 2005, from the Interdepartmental Qualification Commission. From July 2005 to August 2008 he participated in advanced space flight training.

Following the launch of ISS Expedition 23/24 on Soyuz TMA-18 in April 2010, Borisenko was released from his duties on the flight’s backup crew and assigned to the prime crew of Expedition 27/28, as flight engineer on Expedition 27 and commander on Expedition 28. He and his two crewmates, Soyuz commander Aleksandr Samokutyayev of Roscosmos and NASA astronaut Ronald Garan, launched aboard Soyuz TMA-21 from the Baikonur Cosmodrome’s Gagarin’s Start launch pad, at 23:18:20 UTC on April 4, 2011. The launch of Soyuz TMA-21 was dedicated to the 50th anniversary of the first manned space flight by Yuri Gagarin performed in 1961. After 2 days of autonomous flight, the Soyuz TMA-21 spacecraft docked with the International Space Station (ISS) on April 6 at 23:09 UTC, with the crew officially joining the crew of Expedition 27, alongside Russian commander Dmitri Kondratyev, NASA astronaut Catherine Coleman and Italian astronaut Paolo Nespoli.

On 22 May 2011 Kondratyev officially handed over command to Borisenko, with Expedition 27 officially ending with the departure of Soyuz TMA-20, carrying Kondratyev, Coleman and Nespoli back to Earth on 24 May 2011. The Expedition 28 crew would soon be joined by the crew of Soyuz TMA-02M, carrying Russian cosmonaut Sergey Volkov, JAXA astronaut Satoshi Furukawa and NASA astronaut Mike Fossum.

Borisenko launched for a second time on 19 October 2016 aboard Soyuz MS-02 as Flight Engineer, launch was originally scheduled for 23 September although was delayed due to technical issues with the spacecraft.

Borisenko and his two crew mates, Roscosmos cosmonaut Sergey Ryzhikov and NASA astronaut Shane Kimbrough rendezvoused and docked to the ISS on the 21st of October following a two-day rendezvous, and officially joined the Expedition 49 alongside Russian commander Anatoli Ivanishin and flight engineers Takuya Onishi of JAXA and Kathleen Rubins of NASA. Due to month long launch delay Soyuz MS-02 faced, the hand over period was short, Expedition 49 ended on 30 October 2016 when Ivanishin, Onishi and Rubins returned to Earth aboard Soyuz MS-01, leaving the MS-02 trio on board the ISS as Expedition 50 with Kimbrough taking command.

The rest of the Expedition 50 crew joined Borisenko and his two crew mates on 19 November 2016 when Soyuz MS-03, carrying Russian cosmonaut Oleg Novitsky, French astronaut Thomas Pesquet, and NASA astronaut Peggy Whitson docked to the station. During the Expedition the Russian Orbital Segment of the ISS was supposed to welcome the Progress MS-04 uncrewed resupply spacecraft, although the spacecraft failed to reach the ISS due to an issue with the Soyuz-U launch vehicle’s third stage which shutdown sooner than intended. Progress flights quickly resumed during Expedition 50, with the launch of Progress MS-05 in February 2017.

In 2018, in an exposition on space at the Lycée International de Saint-Germain-en-Laye organised by the Russian Section, he lent his suit to participate in the expositon.