A fluorescence polarization immunoassay (FPIA) based on a polyclonal antibody was developed for the determination of melamine in milk. To obtain an antibody with improved sensitivity and specificity, 6-hydrazinyl-1,3,5-triazine-2,4-diamine was coupled to bovine serum albumin and used as the immunogen for the rabbit immunization. Three fluorescein-labeled melamine tracers with different structures and spacer bridges were synthesized. The structural effect of the tracers on the assay characteristics was investigated. 6-(4,6-Diamino-1,3,5-triazin-2-ylamino)-N-(2-(3-(3',6'-dihydroxy-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-5-yl)thioureido)ethyl)hexanamide demonstrated better sensitivity than 5-(2-(4,6-diamino-1,3,5-triazin-2-yl)hydrazinecarbothioamido)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid and 3-(4,6-diamino-1,3,5-triazin-2-ylthio)-N-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-yl)thioureido)ethyl)propanamide. The limit of detection (10% inhibition) of the FPIA was 9.3 ng mL(-1) and the IC(50) (50% inhibition) value was 164.7 ng mL(-1). The antibody in the FPIA showed 21.2% cross-reactivity to the fly-killing insecticide cyromazine, but had no cross-reactivity to other natural structurally related compounds. Recoveries, measured in spiked milk and milk powder samples, ranged from 79.4 to 119.0%. Milk samples fortified with melamine were analyzed by this method and confirmed by high-performance liquid chromatography-mass spectrometry. Excellent recoveries and correlation with spiked levels were observed, suggesting that this immunoassay could be applied to the screening of melamine residues in milk and milk powder after a simple dilution procedure.