A novel protease/EDTA-2Na hydrolysis (PEH) pretreatment method was developed with the combination of EDTA-2Na hydrolysis (EH) and protease hydrolysis (PH) to improve volatile fatty acids (VFAs) production by anaerobic fermentation of waste activated sludge (WAS). Efficiencies of PEH, PH, EH and thermal alkaline hydrolysis (TAH) were compared in releasing SCOD from WAS. Results indicated that physicochemical pretreatment, TAH, had higher SCUD release but less utilization rate of the released SCUD in anaerobic fermentation, biological pretreatments, PH, showed less SCOD release but higher SCUD utilization rate, and PEH could simultaneously obtain higher SCUD release and utilization rate. By TAM, released SCUD could reach 19,273.21 mg/I, but VFAs was only 11,820.36 mg COD/L. By PEH, SCUD and VFAs were 13,628.98 and 12,704.44 mg COD/L, respectively. SCUD released by PEH showed good biodegradability with BOD5/COD of 0.64%, 25.49% higher than that of TAH. By anaerobic fermentation, volatile suspended solids (VSS) of WAS pretreated by PEH could be reduced by 18.50%, but only 6.16% for TAM treated WAS. In PEH process, synergistic effects between protease and EDTA-2Na were observed, in which EDTA-2Na prevented protease from being trapped on the surfaces of sludge floccules and finally greatly improved protease efficiency. Therefore, it is feasible to apply PEH on enhancing VFAs production in anaerobic fermentation of WAS due to its higher and better bio-available carbon release than other pretreatments. (C) 2015 Elsevier B.V. All rights reserved.