In order to improve the efficiency of two-stage anaerobic fermentation system, the circulation process of sludge, composed of the activated sludge online and the activated sludge of the optimal period from storage tanks, was studied. Three factors, including feed concentration, day feeding quantities and sludge recycle ratio, were selected as independent variables, while COD removal rate and biogas production amount were as evaluating indicators. With the quadratic orthogonal rotating combinatorial method, the effects of different parameters on COD removal rate and biogas production amount were analyzed. The results showed that feed concentration, day feeding quantities and sludge recycle ratio were main influencing factors on two evaluating indication, and the mixed sludge recycle ratio had a significant effect. For the anaerobic fermentation system, the optimum parameters of process were feed concentration of 8.21%, day feeding quantities of 7.15L/d and sludge recycle ratio of 30%, respectively.