Protein hydrolysate is critical for the recovery of sludge proteins. Ultrasonic-assisted enzymatic hydrolysis (UEH) is an efficient method for extracting and hydrolysing sludge protein. The process of protein extraction and hydrolysis during UEH and enzymatic hydrolysis (EH) was compared, and it was found that UEH facilitated the extraction of the protein and therefore did not significantly restrict the subsequent hydrolysis of protein as EH did. The extraction of protein conformed to the first-order kinetic equation, and the constants of UEH (k = 1.63 h− 1) were twice those of the EH method. The formation of peptides, an important intermediate product of the proteolytic process, conformed to the tandem kinetic equation, was significantly affected by the dose of enzymes instead of ultrasonic assistance. Further studies on intracellular and extracellular protein extraction and hydrolysis showed that ultrasonic pretreatment effectively reduced the EPS barrier by approximately 50%. Coupled with the synergistic effect of ultrasound and enzyme, the release of intracellular protein was 1.9 times that of EH, which was beneficial to the yield of peptides by removing the restriction in tandem reactions.