Nuclease P1 can hydrolyze nucleic acid into four 5'-mononucleotides, which are widely used as food additives and pharmaceutical intermediates. Nuclease P1 is mainly obtained by microbial fermentation and the low fermentation efficiency of microorganisms for enzyme production is the limit of its application. In order to improve the productivity of nuclease P1, a semi-continuous fermentation of Penicillium citrinum TKZY02 using free-cell was established and the residual sugar, retention volume and air–liquid ratio were optimized. The results indicated that at least 6 fermentation times was performed under the optimum semi-continuous fermentation condition, and the average enzyme activity was up to 518.2 U/mL, which were 16.5% and 9.7% higher than those of batch fermentation and original semi-continuous fermentation, respectively. The average productivity reached 20.9 U/mL/h, which was 42.4% and 21.5% higher than those of batch fermentation and original semi-continuous fermentation, respectively. These findings indicated that Penicillium citrinum TKZY02 was suitable for the production of commercially acceptable levels of nuclease P1 in semi-continuous culture.