A competitive time-resolved fluoroimmunoassay (TR-FIA) was developed for the determination of diethylstilbestrol (DES) residues in chicken liver. Prior to analysis, the residues were extracted from chicken liver with acetonitrile. The assay could be used in a quantitative or qualitative mode. The limit of detection (LOD) was determined to be 0.05 ngg(-1), and the limit of quantification (LOQ) was less than 0.18 ngg(-1). The intraassay variations were less than 10%, and the interassay variations were from 9.8 to 12.7%. The mean recoveries established at six concentration levels varied from 84.3 to 109.6%, and the coefficients of variation were from 8.3 to 12.4%. The results obtained by the TR-FIA and enzyme-linked immunosorbent assay (ELISA) showed a good correlation. The established TR-FIA was validated for the determination of incurred chicken liver and was confirmed by liquid chromatography and tandem mass spectrometry (LC-MS/MS). This proposed technique could be applied to routine residue analysis.