[Objective] Prepare anti-aflatoxin B1 monoclonal antibodies to develop an indirect competitive ELISA (ic-ELISA) applied to detect AFB1 in samples. [Methods] The aflatoxin B1 (AFB1) was conjugated with bovine serum albumin by the carbodiimide method and used to immunize Balb/c mice. Hybridoma cell strains could be obtained by clonal screening and monoclonal antibodies (mAb) could be obtained by inducing ascites in vivo. The subclass and titer of mAb were identificatied by indirect ELISA. After optimizing experimental conditions, a stable ic-ELISA would be developed and applied to detect AFB1 in feed samples. [Results] Four hybridoma cell strains stably secreting specific antibodies to AFB1 were obtained. One of the cell strains 3B9 was chosen to prepare mAb, The mAb belongs to IgG1 with a titer of 1: 204 800. Its cross-reactivity with aflatoxins B2, G1, G2 and M1 was 2.2%, 33.9%, 1.8% and 4.1% respectively. It did not react with ochratoxin A, fumonisin and zearalenone. An indirect-competitive ELISA (ic-ELISA) coated with the mAb had a detection range of 1.04?25.00 μg/L with the detection limit at 1.04 μg/L and the 50% inhibitory concentration (IC50) at 6.03 μg/L. The average recovery rate ranged from 85% to 120% with the variation coefficient less than 10%. [Conclusion] The ic-ELISA has good consistency compared with a commercial kit. We suppose that the ic-ELISA could be used to detect AFB1 from food or feed samples.