Malachite green (MG) is a carcinogenic, teratogenic, and mutagenic chemical that is often illegally used in fishery production. The chemical residues mainly exist in two forms, as MG and its metabolite leucomalachite green (LMG), both of which have severe carcinogenicity, in aquatic products and the environment. Due to the significant structural differences of these two compounds, existing monoclonal antibodies are only able to detect one drug residue in a sample, and cannot accurately reflect the total amount of MG and LMG residues. To date, immunoassay methods for simultaneous detection of MG and LMG residues have rarely been reported owing to the structural differences between the two. Highly specific monoclonal antibodies of MG and LMG were prepared separately, while the indirect competitive enzyme-linked immunosorbent assay (ELISA) for the simultaneous detection of MG and LMG was developed based on the hybrid antibody model. The half maximal inhibitory concentration (IC50) value of the established method on MG and LMG was 3.27 ng/mL, while the limit of detection (LOD) was 0.24 ng/mL and the linear range was 0.62~17.25 ng/mL. Thus, the establishment of the indirect competitive ELISA (icELISA) method is of great significance for ensuring the safety of aquatic food, in addition to providing a reference method to detect two or more residual chemicals simultaneously by the hybrid antibody assay.