The effect of Papaya protease(PAP), Neutral protease(NEP) and Bromelain protease(BRP) on the qualities of biscuit with different dosages were studied on specific volume, texture and color of hard biscuit compared with the addition of Sodium metabisulfite (SMS) and cysteine(CYS), respectively. The results showed that all the 3 proteases could improve the specific volume, texture of biscuit. With the same amount of addition, PAP has the best effect of improving the qualities of biscuit than SMS and CYS. NEP and BRP were weaker than SMS, but better than CYS. It was found that degradation of glutenin macromolecule polymer in gluten was the major factor to weaken the gluten and improve the quality of biscuit for the proteases’ addition after the parameters of dough rheology, flour solvent retention capacity(SRC) and gluten protein molecular weight distribution. were determined. Essential differences between the proteases and the chemical additives in the way to weaken the gluten were found.