CGA-N46 is a small antifungal-derived peptide and consists of the 31st-76th amino acids of the N-terminus of human chromogranin A. Polycistronic expression of recombinant CGA-N46 in Bacillus subtilis DB1342 was used to improve its production, but the yield of CGA-N46 was still low. In the present study, response surface methodology (RSM) was used to optimize culture medium composition and growth conditions of the engineered strain B. subtilis DB1342(p-3N46) for the further increase in CGA-N46 yield. The results of two-level factorial experiments indicated that dextrin and tryptone were significant factors affecting CGA-N46 expression. Central composite design (CCD) was used to determine the ideal conditions of each significant factors. From the results of CCD, the optimal medium composition was predicted to be dextrin 16.6 g/L, tryptone 19.2 g/L, KH2PO4·H2O 6 g/L, pH 6.5. And the optimal culture process indicated inoculation of B. subtilis DB1342(p-3N46) seed culture into fresh culture medium at 5 % (v/v), followed by expression of CGA-N46 for 56 hours at 30 °C induced by 2 % (v/v) sucrose after one hour of shaking culture. To test optimal CGA-N46 peptide expression, the yeast growth inhibition assay was employed and it was found that under optimal culture conditions, CGA-N46 inhibited the growth of Candida albican by 42.17, 30.86 % more than that in the pre-optimization conditions. In summary, RSM can be used to optimize expression conditions of CGA-N46 in engineered strains B. subtilis DB1342(p-3N46).