Abstract: The process of using free cell method produce androstenedione was optimized by response surface methodology. First,the Plackett-Burman experimental design was used to screen out three main factors affecting yield:Phytosterol concentration,hydroxypropyl-β-cyclodextrin concentration,and bacterial cell amount. On this basis,the steepest climbing path method was used to approach the region of maximum response value. Finally,the response surface analysis method was used to determine the interaction between the main factors and the optimal conditions. The results showed that when the phytosterols concentration was 19.41 g/L,the hydroxypropyl-β-cyclodextrinconcentration was 80.14 g/L,the bacterial cell amount was 110.55 g/L,the maximum theoretical yield of androstenedione was 67.25%. After three parallel experiments,the actual average yield was close to the predicted yield.At this time,the androstenedione concentration reached 8.68 g/L,and the production intensity was 2.17 g/(L·d).