In this study, kinetic evaluation of enzymatic hydrolysis of catfish (Pangasius hypophthalmus) by-product meat was conducted. The effects of key factors such as type of proteases (neutrase, papain, and bromelain), enzyme to substrate ratio (E/S), substrate concentration, and extent of hydrolysis on process efficiency, properties and functionalities of hydrolysates were evaluated. The results demonstrated that, for catfish shredded meat, bromelain and papain had higher affinity than neutrase. Kinetics parameters Vm and Km of neutrase, papain, and bromelain were 4.486, 9.060, and 18.249 μM/min and 35.433, 6.582, and 7.034 g/l, respectively. Optimal E/S ratios of neutrase, papain, and bromelain were estimated to be 0.92, 2.76, and 3.31 mg/g. At optimal E/S, the highest possible substrate concentrations were found to be 108.4 g/l for neutrase, 36.2 g/l for papain, and 135.8 g/l for bromelain. Both kinetics study and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that bromelain and papain had higher proteolytic activity toward catfish protein than neutrase. A significant increase in antioxidant activity of hydrolysates was observed with neutrase and papain hydrolysate, but not with bromelain. The results could be helpful for industrial processors to develop an efficient process for catfish by-product valorization.