An indirect, competitive enzyme-linked immunosorbent assay (icELISA) was developed for rapid determination of residual methyltestosterone (MT) in aquatic products. MT was reacted with succinic anhydride to obtain the hapten MT17, which was then coupled to keyhole limpet hemocyanin (KLH) as an immunogen (MT17-KLH). After animal immunization, the polyclonal antibody was successfully obtained and could specifically recognize MT (cross-reactivity with other structurally related chemicals was < 1%). The optimized assay conditions for icELISA were as follows: coating antigen concentration at 100 μg/L; dilution ratio of MT polyclonal antibody at 1:1.2 × 105; 40 min reaction time of antibody; phosphate-buffered saline (PBS) as dilution solution; extraction of aquatic product with acetonitrile, followed by purification by dispersive solid-phase extraction (DSPE) using ethylenediamine-N-propyl (PSA)-bonded sorbent. The half inhibition concentration (IC50) was 3.48 μg/L, while the linear range (IC20~IC80) was 0.77 to 13.06 μg/L. Recoveries from spiked aquatic products were in the range of 81.02% to 103.19%, with relative standard deviation ranging from 4.46% to 13.14%. The results showed good correlation with liquid chromatography-tandem mass spectrometry (R = 0.9971). The method developed in this study is suitable for rapid determination of methyltestosterone residues in aquatic products.