Abstract: With synthesized leuco malachite green-bovine serum albumin （LMG-BSA） as antigen, BALB/c mice was immuned and 1 hybridoma cell line coded 2E6 secreting monoclonal antibody against leueo malachite green （LMG）stably was established by the hybridoma technique. The identification results showed that the number of chromosome of the hybridoma cell reached （99.12±3.6）.The IC50 value of LMG was 20.39 μg/L by competitive inhibition enzyme-linked immunosorbent assay （ciELISA） and the antibody titer inducing mice ascites was 2.3×10^5. The cross reaction between the monoclonal antibody and prohibited hormones and antibacterial drugs seen commonly in aquieuhure was less, which showed that the monoclonal antibody had better sensitivity and specificity.