A fluorescence polarization immunoassay (FPIA) was developed based on a polyclonal antibody for the determination and the qualitative screening analysis of sulfamethazine (SMZ). To optimize the FPIA procedures, three fluorescein-labeled SMZ and sulfamerazine (SMR) were synthesized, and the influence of their structures on the characteristics of FPIA was studied. The optimized FPIA for SMZ had a dynamic range from 5 to 458 ng ml−1 with an IC50 value of 46 ng ml−1 and a detection limit of 1.6 ng ml−1. The specificity of the FPIA was investigated using other 16 sulfonamides, and the cross-reactivity for SMZ, SMR, and sulfamoxol were 100%, 8.6%, and 2.4%, respectively. The kinetics of displacement of SMR-fluorescein isothiocyanate from the immunocomplex by excess of SMZ was also studied.