The invention provides an antibody for testing free gossypol, an ELISA (enzyme-linked immune sorbent assay) method and a kit, and relates to the technical field of analysis of toxic substances in cottonseed meal and feed. Gossypol conjugates with bovine serum albumin through a cross-linking arm to obtain an immunogen, and an immunogen immunized mouse obtains the antibody; according to the ELISA method, firstly, the optimal concentration and test conditions of various reagents used in the ELISA testing method are optimized and determined, and the ELISA testing method for gossypol is developed; the pretreatment condition of a to-be-tested sample is determined, and the to-be-tested sample is tested with the built ELISA method. The kit comprises an elisa plate coated with a coating antigen, standard gossypol bis-adduct solutions with different concentrations and an antibody working solution. According to the antibody, the ELISA method and the kit, the specificity and the appetency of the antibody in recognition of a gossypol derivative are improved, and the test sensitivity of the ELISA method and the test sensitivity of the kit are improved.