Klitschko, who also holds the WBO, IBO and WBA titles, knocked out Pulev with a fierce left hook in the fifth round for his 63th win in 66 fights.
Pulev, who had won his previous 20 fights, protested several times about being subjected to rigorous drug-testing, saying that Klitschko had not been tested at all before the bout on November 15.
He repeatedly called for his 38-year-old opponent to be tested but the Ukrainian refused.
“You can respect him, he’s a world champion,” said 33-year-old Pulev, who became the IBF international champion in 2011 and European champion in 2012.
Pulev failed to turn up to the news conference before the fight in Hamburg after being informed that only three people from his camp could be present.
The Bulgarian also accused Klitschko in August of feigning injury to avoid taking him on in September. Klitschko postponed the fight, saying he had torn bicep muscles during training in Austria.