Short-chain fatty acids (SCFAs), the carbon source of biological nutrient removal, can be produced by waste activated sludge (WAS) anaerobic fermentation. To get more SCFAs from sludge, most studies in literature focused on the mechanical process control or the structure of microbial community; little attention has been paid to the key microorganisms and their function related to SCFA generation. In this study, a different sludge pretreated method, i.e., pretreating sludge by proteinase K for 2 days followed by pretreating at pH 10 for 4 days, is reported, by which the proportion of Clostridiales was increased and SCFA generation was enhanced. First, the effects of different proteinase K concentrations and initial pH on sludge hydrolysis and SCFA generation were investigated. The optimal conditions showed the highest SCFA generation (352.91 mg COD per gram of volatile suspended solids), which was 2.89-fold of the blank (un-pretreated). Further, the new biological pretreatment process led to the conversion of other SCFAs to acetic acid. Acetic acid accounted for 60.8 % of total SCFAs with the new biological pretreatment process compared with 44.9 % in the blank test. Then, the investigation on the key microorganisms related to SCFA production with 16S rRNA gene clone library and fluorescence in situ hybridization (FISH) indicated that there were much greater active Clostridiales when SCFAs were generated with the proteinase K and pH 10 pretreated sludge. Further, the mechanisms for the optimal conditions significantly enhancing SCFA generation were investigated. It was found that pretreating sludge by proteinase K and pH 10 caused the greatest key enzyme activities, organic consumption, and inhibition of methane generation. Graphical abstract A new biological process for short-chain fatty acid generation from waste activated sludge improved by Clostridiales enhancement.