The invention relates to a culture method of producing bacteria of penicillin acylase and belongs to the technical field of catalyst production. The culture method of the producing bacteria of the penicillin acylase comprises steps as follows: (1) preparation of a culture medium; (2) activation of strains; (3) preparation of a strain culture plate; (4) dilution of a seed solution; (5) coating of the seed solution and selection; (6) culture of the strains; (7) pre-screening with shake flasks; (8) re-screening with shake flasks. With the adoption of the culture method of the producing bacteria of the penicillin acylase, the high-activity producing bacteria of the penicillin acylase can be cultured, and the penicillin acylase produced by the cultured producing bacteria is high in activity.