The present invention provides a preparation method of a lactic acid bacterium protein raw material with a high content of small peptide and application thereof, and the method is performed according to the following steps: adjusting the pH of a stock solution of lactic acid bacterium liquid to 7 to 8; adding 0.05% to 0.5% of pancreatin to the stock solution of lactic acid bacterium liquid to obtain a mixed solution A; performing enzymolysis on the mixed solution A obtained in the step b for 2 to 6 hours, wherein the enzymolysis temperature is 50 to 60 DEG C; heating the product obtained in the step c, inactivating the pancreatin, and performing spray drying to obtain the protein raw material with a high content of small peptide. According to the invention, waste lactic acid bacterium liquid obtained during production of lactobacillin is utilized to produce the lactic acid bacterium protein raw material, which belongs to waste utilization and can greatly relieve economic pressure of a feed manufacturer. For directed enzymolysis provided by the present invention, enzymolysis conditions are neutral and slightly alkaline (basically, no adjustment is necessary), enzymolysis time is short and 2 to 5 hours, operation time is short, and enzymolysis efficiency is high.