He made his debut during the 1990–91 season in a match against his future club, Olympiacos. In his first years as a Panathinaikos player he was a back-up for Józef Wandzik. In 1995, he played five games as Panathinaikos won the championship and three as they retained it the following year. However, it wasn’t until the 1997–98 season that he established himself as a regular goalkeeper at the age of 26. In the 2002 Champions league, he helped the club reach the quarter-finals but, in the 2003–04 season, he lost his place to Konstantinos Chalkias when his contract negotiations broke down.

Nikopolidis made his debut for the Greece national football team on 18 August 1999 against El Salvador.

On 15 June 2008, Nikopolidis announced his retirement from international football after Euro 2008, claiming that he had made this decision before the tournament started, and also stating that it is about time for a major change in the Greek national squad.

He also took part in the international match “8th Match Against Poverty”, on 14 December 2010, in Karaiskakis Stadium in Greece, as the UNDP team’s goalkeeper.

In January 2011, IFFHS released a list with the best goalkeepers of the first decade, and Nikopolidis was in. He was also listed as one of the best goalkeepers of the years 1987 to 2011.

On 20 March 2011, Olympiacos won the championship in a 6–0 win against rivals AEK Athens F.C., in Karaiskakis Stadium.

He made his final appearance on 17 April 2011 in a home 6–0 victory against Larissa F.C. in Karaiskakis Stadium. As a final curtain call, he was replaced by Balázs Megyeri in the last minutes, so he could wave goodbye to the Olympiacos fans.

After retirement, Nikopolidis became the assistant manager of Olympiacos in 2012.

He was promoted to manager in January 2013 on an interim basis following the dismissal of Leonardo Jardim. After the arrival of Míchel, he remained in the team as his assistant.

On 1 October 2015, he was named as manager of the Greece national under-21 football team.