N-Acetyl--D-glucosaminidase(EC 188.8.131.52, NAGase), hydrolyzing the oligomers of N-Acetyl--D-glucosamine(NAG) into monomer, is correlated with animal reproduction. In the current study, we investigated the effect of two common drugs(CuSO4 and ZnSO4) on NAGase from spermary of Nile tilapia(Oreochromis niloticus). The results showed that the IC50 of CuSO4 and ZnSO4 were(1.230.05) mmol/L and(0.280.02) mmol/L, respectively. Both CuSO4 and ZnSO4 regulated the conformation of tilapia NAGase, which affected the fluorescence emission of the enzyme. The inhibitory activities of these two drugs were reversible, and the inhibition type of CuSO4 was noncompetitive but ZnSO4 was competitive. Both CuSO4 and ZnSO4 had significant influences on the thermal and pH stability of the enzyme. The results contribute to the use and control of CuSO4 and ZnSO4 in tilapia culture.