Abstract: Cassava starch residue, as a promising raw material for biogas production, is rich in cellulose. Due to high water content and scattered solid particles, it is difficult for solid cassava starch residue to ferment and it had to experience hydrolysis pretreatment as raw material for biogas production. The hydrolysate obtained by various hydrolysis pretreatment has major impact on the rate of acid production and acid distribution in subsequent acidification. The hydrolysate of cassava starch residue, pretreated by hot water and amylase respectively, was biological fermented. The results showed that, for the biological acidification of hydrolysate using α-amylase, hydrolyzed for 20 min by hot water at 150 ℃ and hydrolyzed for 45 min by hot water at 150 ℃, the maximum acid yield increased respectively to 5 277, 6 209, 4 734 mg/L from 4 558 mg/L and the corresponding time shortened to 30 h, 48 h, 24 h from 96 h, compared with the condition of cassava starch residue only. Besides, an optimum volatile fatty acids (VFAs) distribution was obtained in 24 h for the three kinds of hydrolysate, and the sum of acetic acid and butyric acid was about 90% of total VFAs. According to the acid produced rate and acid distribution, an optimum dissolving method was that the cassava starch residue was hydrolyzed for 20 min by hot water at 150 ℃ and the subsequent biological acidification time was 24 h.