The 6-foot-11 Croatian broke the previous record of 49, established by Richard Krajicek in a 1999 quarterfinal loss.
Karlovic, who is seeded 21st, beat Yen-hsun Lu of Taiwan 4-6, 7-6 (4), 6-7 (4), 7-6 (5), 7-5 on Tuesday, holding in 28 of his 29 service games.
Karlovic finished with 10 double-faults.
He hit 22 aces in the second set alone — twice as many as Andy Murray produced in his entire three-set victory Tuesday night.
The record for most aces in a match is 113, set by John Isner during the longest match in tennis history, his victory over Nicolas Mahut that ended 70-68 in the fifth set during Wimbledon's first round in 2010.