Markus John was born on February 17, 1962, in Duisburg. In 1981, he began training at the Zinner-Studio acting school in Munich. After graduating in 1984, he worked at several prestigious theaters, including the Bavarian State Theater in Munich, the Stadttheater Bern, the Thalia Theater in Hamburg, the Berliner Ensemble, and the Deutsches Theater Berlin. In 1997, he joined the ensemble of the Schauspiel Köln (Cologne Theater).

In addition to his extensive stage work, John began occasionally appearing in television productions in the late 1990s. He mostly played smaller supporting roles in crime series such as "Balko," "Tatort," "Die Kumpel," and "Die Wache." He also appeared on the big screen in small parts of the character study "Narren" ("Fools", 2003) and Sherry Hormann's comedy "Männer wie wir" ("Guys and Balls", 2004), in which he played a member of a gay soccer team.

In the 2009/2010 season, John moved from Berlin to join the ensemble of the Deutsches Schauspielhaus Hamburg. In 2013, he was awarded the Rolf Mares Prize for his title role in "Oedipus." As curator, director, and performer, he realized "Foxi, Jussuf, Edeltraud" in 2012, a one-person program in which the entire text is based on interviews John conducted with people he chose at random and did not know beforehand. In the 2014/15 season, he had a guest appearance at the Schauspiel Hannover, in a production of Schiller's "Maria Stuart".

From the 2010s onwards, John's TV roles became more prominent. He played the father of a murder victim in the Dortmund "Tatort" episode “Eine andere Welt” (2013) and a repeat sex offender in the Rostock "Polizeiruf 110" episode “Angst heiligt die Mittel” (2016). In the six-part sitcom "Ellerbeck" (2015), he had a leading role as a city councilor of a fictional small town. In cinema, he was part of the ensemble cast of Sönke Wortmann's film adaptation of Frank Goosen's "Sommerfest" (2017).  

In 2019, John played the lead role of a family man in the small-town social drama "Eher fliegen hier Ufos" (TV), which was not broadcast until the end of 2023 – and earned John a Deutscher Schauspielpreis. In 2022 he took on a permanent leading role as a police inspector in the series "WaPo Duisburg".

John's main field of activity, however, remained theater. In 2023, he was awarded the Bauturm Art Prize at the Cologne Theater im Bauturm, where he regularly performed with his program "Foxi, Jussuf, Edeltraud."

His first leading cinema role was in the tragicomedy "Seid einfach, wie ihr seid" ("Act Natural") (2022). In it, he played the father of a film student who wants to make her thesis film about the reunion of her long-separated parents. "Act Natural" premiered at the Max Ophüls Prize Film Festival in January 2023 and was released in theaters in fall 2025.