Kline was born in Wilkes-Barre, a small coal-mining community in Eastern Pennsylvania. When he was seven years old, Kline’s father committed suicide. During his youth he moved to Lehighton, Pennsylvania and graduated from Lehighton High School. His mother later remarried and sent him to Girard College, an academy in Philadelphia for fatherless boys. After graduation from high school, Kline studied art at Boston University from 1931 to 1935, then spent a year in England attending the Heatherley School of Fine Art in London. During this time, he met his future wife, Elizabeth V. Parsons, a British ballet dancer. She returned to the United States with Kline in 1938.

Upon his return to the country, Kline worked as a designer for a department store in New York state. He then moved to New York City in 1939 and worked for a scenic designer. It was during this time in New York that Kline developed his artistic techniques and gained recognition as a significant artist.

Kline’s artistic training focused on traditional illustrating and drafting. During the late 1930s and early 1940s Kline worked figuratively, painting landscapes and cityscapes in addition to commissioned portraits and murals. His individual style can be first seen in the mural series Hot Jazz, which he painted for the Bleecker Street Tavern in Greenwich Village in 1940.

In 1946 the Lehighton, Pennsylvania Post of the American Legion commissioned Kline to do a large canvas depicting the town where he had attended high school. The work now known as The Lehighton Mural was acquired from the American legion post in 2016 by the Allentown Art Museum in Allentown, Pennsylvania and is today on permanent exhibition there.

It is widely believed that Kline’s most recognizable style derived from a suggestion made to him by his friend and creative influence, Willem de Kooning. De Kooning’s wife Elaine gave a romanticized account of the event, claiming that, in 1948, de Kooning advised an artistically frustrated Kline to project a sketch onto the wall of his studio, using a Bell–Opticon projector. Kline described the projection as such:

Kline had his breakthrough show at the Charles Egan Gallery in 1950, and he participated in the 9th Street Art Exhibition the following year. In 1958 he was included in the Museum of Modern Art’s major exhibition, “The New American Painting”, which toured eight European cities. In the decade before his death, his work was included in numerous international exhibitions, including the Venice Biennale (1956, 1960); Documenta, Kassel, West Germany (1959); São Paulo Biennial (1957); and Whitney Annuals and Biennials (1952, 1953, 1955, 1961). The Washington Gallery of Modern Art, Washington, D.C., organized a memorial exhibition (1962). Major monographic exhibitions have also been held at the Whitney Museum of American Art, New York (1968); Phillips Collection, Washington, D.C. (1979); Cincinnati Art Museum, traveling to San Francisco Museum of Modern Art and Pennsylvania Academy of Fine Arts (1985); Menil Collection, Houston (1994); Fundació Antoni Tàpies, Barcelona (1994); and Castello di Rivoli, Museo d’arte contemporanea, Italy (2004).

In the later 1950s, in such paintings as Requiem (1958), Kline began experimenting with more complex chiaroscuro instead of focusing on a strict monochromatic palette. Then in 1958, he reintroduced the use of color in his work through colorful accents in his black and white paintings. This exploration back to color-use was still in development when Kline died in 1962.

He later taught at a number of institutions including Black Mountain College in North Carolina and the Pratt Institute in Brooklyn. He spent summers from 1956 to 1962 painting in Provincetown, Massachusetts, and died in 1962 in New York City of a rheumatic heart disease, ten days before his 52nd birthday.

An early work, UNTITLED, from 1940 (of an interior room) was purchased from Sotheby’s in 1995 by a private collector for $21,850. This early piece helps to define his early phase, before his transformation from a realist painter to a groundbreaking abstract expressionist. The painting’s bold brushstrokes prefigure the epic black abstraction of his breakthrough style.

In 2012 San Francisco financier George R. Roberts sold a nearly ten-foot (3 m) wide, untitled black-and-white work from 1957 at Christie’s, New York. The painting went to a telephone bidder for $36 million, or $40.4 million with fees (Christie’s guaranteed the seller Robert Mnuchin an undisclosed minimum), a record price for the artist at auction and more than six times the previous record, which was set in 2005 when Christie’s sold Crow Dancer (1958) for $6.4 million.