Papain incorporated films were developed as novel edible packaging for meat tenderization. Pregelatinized high dissolution (HD) and low dissolution (LD) cassava starch with different water solubilities were incorporated with papain. The edible films produced were characterized for morphology, mechanical relaxation and mechanical and barrier properties. Papain interacted with and modified the O-H bond through hydrogen bonding and disrupted the ordered structure of starch, while increasing the intensity of mechanical relaxation. LD produced more homogeneous microstructures than HD films containing papain by preventing aggregation. Interaction between starch and papain stabilized the film structure, reducing water sensitivity and solubility. Films containing papain at 5% to 15% showed improved tenderness of beef, with reduced Warner-Bratzler shear values and hardness after incubation for 30 min. HD films gave higher tenderization due to fast dissolution and release of enzymes on the meat surface, while increased papain in films improved the degree of tenderness and protein conformation in packaged beef.