Objective To prepare and identify rabbit anti-breakpoint cluster region-Abelson leukemia virus oncogene (BCR-ABL) b3a2 subtype polyclonal antibody. Methods A peptide containing the fusion sequence of the b3a2 subtype BCR-ABL fusion protein was designed and synthesized with the purity higher than 90%. The fusion polypeptide was coupled to Keyhole Limpet hemocyanin (KLH) and used to immune New Zealand rabbits. Antiserum was purified after multiple immunizations, in addition to using the b3a2 subtype fusion polypeptide for affinity purification. Peptides harboring only BCR or c-ABL amino acid sequences were also synthesized and used to purify the antibody in the secondary purification. The antibody that only bound to part of the epitope was absorbed and removed. ELISA and Western blotting were performed to determine the antibody titer and specificity. Results The rabbit serum background was low before immunization. The titer of the polyclonal antibody reached 1:32 000 after immunization, which met the experimental requirements. Western blotting showed that the antibody could specifically recognize the b3a2 subtype fusion protein of BCR-ABL. Conclusion The experiment has prepared the specific rabbit polyclonal antibody against BCR-ABL b3a2 subtype.