To investigate the antigenic epitopes of phospholipase A2 (PLA2) from Deinagkistrodon acutus venom and to prepare specific antibodies against this enzyme, the amino acid sequence of PLA2 was analyzed using the IEDB-Bepipred 2.0 online server for linear B-cell epitope prediction. The identified antigenic epitopes were cloned into the expression vector pET-28a(+) for prokaryotic expression, and the resulting recombinant protein was purified. The purified recombinant protein was used to immunize mice (Mus musculus) for antibody production. Enzyme-linked immunosorbent assay (ELISA) and Western blot were employed to evaluate the antibody titers in mouse sera and their binding activity against PLA2 from D. acutus venom, as well as to assess the inhibitory effect of the antibodies on PLA2 enzymatic activity. Six antigenic epitope sequences of PLA2 were successfully predicted, and the corresponding recombinant protein, with a relative molecular weight of approximately 16.5 kDa, was expressed. ELISA and Western blot analyses showed that mouse sera exhibited antibody titers exceeding 1∶256 and strongly bound to PLA2 proteins with relative molecular weights of 14~16 kDa in D. acutus venom. In vitro enzymatic activity assays demonstrated that 10 μL of immunized serum reduced the activity of 6 μg PLA2 from 100% to 62.2%. These findings indicate that the antigenic epitopes designed based on D. acutus venom PLA2 effectively stimulated an immune response in mice, resulting in the successful preparation of serum antibodies capable of neutralizing PLA2 from D. acutus venom.