Abstract: To further improve the yield of pectinase, the fermentation medium and fermentation conditions for pectinase production by Bacillus subtilis Z-5 were optimized using the response surface methodology, and the optimized pectinase was applied for the clarification of pear juice. Based on the single-factor experiments, the Plackett-Burman test design was applied to screen out three significant factors affecting enzyme production: Yeast powder content, pH and incubation time, and then combined with the response surface design method to determine the optimal fermentation conditions. The results showed that the optimal fermentation medium composition was 10 g/L pectin, 7.99 g/L yeast powder and 0.5 g/L MgSO₄·7H₂O, the optimal fermentation conditions were initial pH5.66, fermentation temperature 37 ℃, incubation time 72 h, inoculum 6% (v/v), liquid volume 50/250 mL and substrate concentration 10 g/L. On the basis of this, the enzymatic activity of pectinase produced by B. subtilis Z-5 was increased from 879.00 U/mL to 1549.62 U/mL, which was 1.76 times higher than the unoptimised one. In pear juice clarification experiments, it was shown that the optimum addition of pectinase was 4 mL, the optimum enzymatic time was 2.5 h, the optimum enzymatic temperature was 65 ℃ and the optimum enzymatic pH was 6.0, the maximum light transmission rate was 79.77%. Compared with the commercial enzyme, the homemade pectinase is a compound enzyme with multiple enzyme systems, which can act on other components such as protein and starch contained in the pear juice, making the pear juice more clarified. The results of this study have provided theoretical support for the industrial production and application of pectinase.