Magnetite nanospheres was prepared and used to fabricate a modified carbon paste electrode as xanthine biosensor and applied to determine inhibition kinetics of Syzygium polyanthum extract toward xanthine oxidase (XO). The magnetite nanospheres was characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). Electrochemical behavior of xanthine was investigated by immobilizing the XO on the surface of carbon paste electrode (CPE), modified magnetite nanospheres-CPE (MCPE), and modified 2,3-dimethoxy-5-metil-1,4-benzoquinone (Q₀)-CPE (QCPE) employing cyclic voltammetry (CV) technique. The result showed that the MCPE was the best electrode to determine the analytical performance under optimum condition, based on lower limit of detection (0.005 mM), wider linearity range (0.01-1 mM with R²= 99.24%), and higher sensitivity (5.16 µM^-1) than the two oter electrodes (CPE and QCPE). The MCPE successfully improve the analytical performance on xanthine biosensor and to be applied for determining inhibition kinetics of S. polyanthum extract. Inhibition kinetics of the extract has caused increase of K_M and V_MAX (I_MAX) constant. Based on the result, the type of inhibition kinetics was a competitive inhibition.