The invention provides a lipase activity determination and performance evaluation method taking fat-and-oil-enriched hide powder as a substrate. The method comprises the steps: preparing a fat-and-oil-enriched hide powder substrate from animal skin, which serves as a raw material; enabling lipase to be in contact with fat and oil adsorbed by the hide powder in an aqueous medium, catalyzing the fat and oil in the hide powder to hydrolyze so as to produce fatty acids, carrying out a reaction under certain conditions, terminating an enzymatic reaction simultaneously with salts and acids, converting salts of fatty acids in bath liquid into fatty acids, and enabling the fat and oil and the fatty acids to be completely adsorbed by the hide powder; completely extracting the produced fatty acids from the hide powder with a solvent, and characterizing the activity and performance of lipase through determining the fatty acid yield of unit time. According to the method, the lipase activity determination and lipase performance evaluation can be carried out in a relatively wide pH range, the substrate and reaction conditions used are close to acting conditions of the lipase to intracutaneous fat and oil in an animal skin treatment process, and determination results have a guiding significance in lipase selection and application conditions in lipase application fields such as leather making, fur coat making, glue making and collagen extracting which adopt the animal skin as the raw material.