The concentration of glycocholic acid (GCA) in urine and blood is an important biomarker for liver cancer. Monitoring of GCA depends to a large extent on the availability of appropriate analytical techniques. In this work, based on the immobilization of GCA-OVA onto the sensor chip surface, a label-free competitive inhibition immunoassay for the determination of GCA with the surface plasmon resonance (SPR) technique was developed. The proposed SPR immunosensor is simple to prepare, recyclable and exhibits excellent sensitivity to GCA (a linear range of 13.3-119.4 ng mL-1 and a limit of detection (LOD) of 2.5 ng mL-1), which was 14 times lower than that of the traditional immunoassay. Excellent recoveries and correlation between these two methods were observed (R2 = 0.995). Hence, it can be proved that the SPR immunosensor could be used to achieve rapid and sensitive quantitative detection of GCA in real urine samples and meet clinical needs.