Milk protein hydrolysates are common in infant formula, but some of them retain allergenicity due to incomplete hydrolysis of the epitopes for milk allergens. This study explored a method for screening proteases that could specifically hydrolyze the epitope of alpha(s1)-casein allergen. Firstly, the alpha(s1)-casein epitope AA83-105 was synthesized by the solid-phase synthesis method. Then, after purification and identification, the complete antigen was prepared through coupling with bovine serum albumin (BSA) and was used to raise monoclonal antibodies (mAb) in BALB/c mice. Additionally, an indirect competitive-enzyme-linked immunosorbent assay (icELISA) was established. The mAb raised against oco-casein protein was used as a control. The results showed that the purity of the synthetic epitope was >90%, and the coupling rate with BSA was 6.31. The mAb subtype is IgG(1), with a titer of 1:320,000. The mAb reacted specifically with alpha(s1)-casein but did not cross-react with soybean protein. The linear regression equation of the competitive inhibition curve was y = -9.22x + 100.78 (R-2 = 0.9891). The detection limit of icELISA method was more sensitive, and the method showed good accuracy and repeatability. The amounts of antigen residues in papain protease hydrolysates were relatively small, and the epitope fragment was detected in papain hydrolysate via mass spectrometry. This study provides ideas and methods for screening proteases that specifically hydrolyze the epitopes of milk allergens and also provides a superior foundation for the development of an advanced hypoallergenic formula.