In the present work, biodegradation behaviors compared commercial polyurethane to synthesized polyurethane in different enzyme solutions. Two quantitative methods have been used to follow the biodegradation. Enzymes were esterase and protease in the hydrolase class; enzyme solutions were prepared one by one or by combining as enzyme cocktail solutions. Esterase affect ester bonds whereas protease enzymes affect urethane and urea linkages. Thus, effects of enzyme cocktail solutions were also researched in this study. It is a new way for in vitro degradation of polyurethane samples by enzyme cocktail solutions. 1cm×1cm of polyurethane films were put in a tube and treated by different combinations of these mentioned enzymes. The tubes were placed in a shaker and stirred at 120 rpm under the reaction condition of 37ºC and pH 7.4. In order to keep enzymatic activity stable, enzyme solutions were prepared again periodically. All samples were characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. The bond strength of chains and morphological changes were recorded during the period of biodegradation. The results showed that protease enzyme solutions could erode polymer films, but esterase enzyme solution could not be so effective alone. In addition, enzyme cocktail solutions showed that enzymes could compete with each other, so one enzyme could suppress the activity of another enzyme.