Abstract: An enzyme-linked immunosorbent assay(ELISA)for detecting the residue of malachite green(MG)and its metabolite leucomalachite green(LMG)in aquatic seedlings was established in the present study.The pretreatment process was optimized by using acetonitrile as the extractant,ultrasonic extraction instead of vortex oscillation extraction,saturated n-hexane with pure water,and marking the centrifugal tube with red marker.The results showed that MG content had good linear in the range of 0.050 0~4.50 ng·mL^-1,which the correlation coefficients was R²=0.997 0,and the detection limit was 0.075 0 μg·kg^-1.The average recoveries ranged from 79.7%~104.5% at the spiked level of 0.075 0,0.150 and 0.750 μg·kg^-1 in the seedlings of Penaeus chinensis,Portunus trituberculatus,Stichopus japonicus and Paralichthys olivaceus.The relative standard deviations(RSD)was 3.3%~7.9%.The results showed that this method was accurate,rapid and cheap,which would be suitable for periodic spot check of MG & LMG residues in different aquatic seedlings substrate.