To improve volatile fatty acids (VFAs) accumulation from hydrolysis and acidification of excess sludge (ES), alkali-tolerant bacteria capable of protease-producing were isolated from ES. A mixed microbial consortium capable of protease-producing was constructed by the isolated bacterial strains. The mixed microbial consortium was inoculated into the different fermentation periods of ES to investigate their effects on soluble organic compounds and VFAs accumulation from ES under alkaline conditions (pH 10.0). The optimal condition for VFAs accumulation from ES was investigated. The results showed that two alkali-tolerant bacterial strains capable of protease-producing were isolated from ES and constructed as a mixed microbial consortium. The soluble organic compounds concentrations and VFAs accumulation were improved significantly after the mixed microbial consortium was inoculated at the initial fermentation, and the start-up phase was shortened by 2 days. On the 8th day of fermentation, the concentrations of soluble protein and total VFAs reached their peak values, and were 1.25 times and 1.41 times higher as compared to the corresponding values from non-inoculated samples, and accounted for 29.87% and 44.54% of total SCOD concentration, respectively. Acetic and propionic acids were the most prevalent VFAs (account for 50.69% and 18.19%, respectively).