A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of the hexoestrol (HES). Polyclonal rabbit antisera, raised against protein conjugate hexoestrol-mono-carboxyl-propyl-ethyl-bovine-serum-albumin (HES-MCPE-BSA), were utilized in immobilized antibody-based and competitive immunoassays. Assay conditions, including concentrations of antisera and Horseradish peroxidase (HRP)-HES were optimized. The effect of incubation time, surfactant concentration, ionic strength and pH of the medium were also investigated. The typical calibration curve gave an average IC50 value of 2.4 ng/ml, calibration range from 0.2 ng/ml to 30.5 ng/ml and a detection limit of 0.07 ng/ml. The specificity of the assay was tested against HES structurally related compounds, and the assay proved highly selective for HES. Assay performance was validated by using spiked pork and liver tissues samples. Moreover, it was compared with liquid chromatography-tandem mass spectrometry. The ion pair for quantification of HES was 269.4/134, and linear equation of HES was Y=0.2148X-0.0374 (r=0.9993). The two analytical methods can be applied to monitor HES and other steroid residues in edible foods.