An amperometric biosensor for the detection of creatine was designed, based on carbon paste electrode modified with Fe3O4 nanoparticles. Electron transfer properties of unmodified and Fe3O4-nanoparticles-modified carbon paste electrodes were investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) methods. Fe3O4 nanoparticles increased the surface area and electric conductivity of the electrode, thus enhancing the sensitivity of the electrode. Optimum pH, buffer concentration, working potential and enzyme loading were selected as 7.0; 0.05 mol L-1; + 0.30 V and 2.0 Unit creatinase (CI), 1.0 Unit sarcosine oxidase (SO), respectively. The purposed biosensor exhibited linear response from 2.0 x 10(-7) mol L-1 to 3.8 x 10(-6) mol L-1 and from 9.0 x 10(-6) mol L-1 to 1.2 x 10(-4) mol L-1 with a detection limit of 2.0 x 10(-7) mol L-1. Biosensor was used for determination of creatine in commercial creatine powder samples and showed a good sensing performance.