During the anaerobic digestion of dewatered sludge, polyacrylamide (PAM), a chemical conditioner, can usually be consumed as a carbon and nitrogen source along with other organic matter (e.g., proteins and carbohydrates in the sludge). However, a significant accumulation of acrylamide monomers (AMs) was observed during the PAM biodegradation process. To improve the anaerobic hydrolysis of PAM, especially the amide hydrolysis process, and to avoid the generation of the intermediate product AM, a new strategy is reported herein that uses an initial pH of 9, 200 mg COD/L of PAM and a fermentation time of 17 d. First, response surface methodology (RSM) was applied to optimize PAM removal in the anaerobic digestion of the sludge. The biological hydrolysis of PAM reached 86.64% under the optimal conditions obtained from the RSM. Then, the mechanisms for the optimized parameters that significantly improved the biological hydrolysis of PAM were investigated by the synergistic effect of the main organic compounds in the sludge, the floc size distribution, and the enzymatic activities. Finally, semi-continuous-flow experiments for a microbial community study were investigated based on the determination of key microorganisms involved in the biological hydrolysis of PAM.