Malachite green(MG), a dye, is an antifungal agent that has been used to treat and prevent fish diseases. It is metabolized into reduced leucomalachite green forms(LMG) that may reside in fish muscles for a long period, thus being harmful to human health. The aim of this study was to develop a competitive and direct enzyme-linked immunosorbent assay(ELISA) to detect MG and LMG specifically. The monoclonal antibody(m Ab) to LMG was generated using a hybridoma technique. The obtained m Ab showed good cross-reactivity(CR) to malachite green(MG), but not to crystal violet(CV) and Brilliant Green(BG). The m Ab was used to develop a fast detecting ELISA of MG and LMG in fish. By introducing the conjugation LMG-HRP, the detection capability was 0.37 ng m L-1 for MG and LMG. The mean recovery from spiked grass carp tissues ranged from 76.2% to 82.9% and the coefficients of variation varied between 1.8% and 7.5%. The stable and efficient monoclonal cell line obtained is a sustainable source of sensitive and specific antibody to MG and LMG. Malachite green (MG), a dye, is an antifungal agent that has been used to treat and prevent fish diseases. It is metabolized into reduced leucomalachite green forms (LMG) that may reside in fish muscles for a long period, was being to human health. The aim of this study was to develop a competitive and direct enzyme-linked immunosorbent assay (ELISA) to detect MG and LMG specifically. The monoclonal antibody (m Ab) to LMG was generated using a hybridoma technique. The obtained m Ab showed good cross-reactivity (CR) to malachite green (MG), but not to crystal violet (CV) and Brilliant Green (BG). The m Ab was used to develop a fast detecting ELISA of MG and LMG in fish. the conjugation of LMG-HRP, the detection capability was 0.37 ng m L-1 for MG and LMG. The mean recovery from 76.2% to 82.9% and the coefficients of variation varied between 1.8% and 7.5%. stable and efficient monoclonal cell line obtained is a sustainable source of sensitive and specific antibody to MG and LMG.