Poly calcein modified glassy carbon electrode (PCA/GCE) has been fabricated by simple electropolymerization method. The electrochemical behaviors of uric acid (UA), xanthine (XA) and hypoxanthine (HX) at PCA/GCE were studied by cyclic voltammetry and linear sweep voltammetry. The results showed that PCA/GCE displays not only electrocatalytic activity toward the electrochemical oxidations of UA, XA and HX, but also good reproducibility, stability and long lifetime. Under the optimum conditions, the linear concentration dependences of LSV peak current responses were observed for UA, XA and HX in the concentration ranges of 0.2-80, 0.2-80 and 0.5-90 μM with the detection limits of 0.12, 0.12 and 0.3 μM, respectively. Moreover, PCA/GCE was applied to simultaneous detection of UA, XA and HX in human urine samples with satisfactory results.