In this paper, a simple, rapid and high-throughput fluorescence polarization immunoassay (FPIA) based on polyclonal antibodies (PAb) is described for the determination of glycocholic acid (GCA) in human urine. Three fluorescein-labeled GCA (tracers) with different structures and spacer bridges were synthesized and purified by thin-layer chromatography (TLC). The structure effect of tracers on the assay was investigated and the sensitivity of best tracer in the optimized FPIA demonstrated an IC50 value of 306ng/mL. The working range of FPIA was 36 ˜ 2 600ng/mL and the limit of detection (LOD) was 9ng/mL. The developed FPIA was time-saving that could be completed within 10min. Human urine samples spiked with GCA were analyzed by this method, followed by confirmation with commercial enzyme immunoassay analysis (EIA). Excellent recoveries and correlation between these two methods were observed (R2=0.996), suggesting the developed FPIA could be applied to screening of GCA in human urine samples without complicated cleanup.