A new monoclonal antibody (mAb) against cephalexin (CEX), named 1F3, was produced by immunizing mice with immunogen CEX–keyhole limpet hemocyanin. Two fluorescein-labeled tracers (CEX–fluorescein isothiocyanate (FITC) and cefadroxil (CDX)–FITC) were synthesized and purified by thin-layer chromatography. A homogeneous fluorescence polarization immunoassay (FPIA) based on the new mAb and the selected tracer for the simultaneous determination of CEX and CDX was developed and optimized. The optimized FPIA provided a limit of detection of 1.8 and 2.3 ng ml−1 for CEX and CDX and exhibited cross-reactivities of 100 and 63.16 % for CEX and CDX, respectively, and <1 % for other 11 cephalosporins. Recoveries from fortified milk were ranged from 82 to 125 % with coefficient of variations of 3.2–14.2 %. The results indicated that the homogeneous FPIA we developed was enough sensitive to rapidly monitor CEX and CDX in milk.