Creatinine deiminase (CD) immobilized on Nafion (R)-nanostructured polyaniline (nsPANi) composite film prepared by the cyclic voltammetry (CV) is used as sensing material to measuring the concentration of creatinine in the aqueous solution. The effect of cycle number, scanning rate, temperature and the concentrations of hydrochloric acid and aniline for preparing composite sensing film on the sensitivity of amperometric creatinine biosensor are systematically investigated. The surface morphologies and the properties of as-prepared Nafion (R)-nsPANi composite film are analyzed by FESEM and FTIR, respectively. The maximum sensitivities for monitoring NH4+ based on Nafion (R)-nsPANi composite film are obtained to be 1647 and 376 mu A mM(-1) in the concentration ranges of 0.005-0.1 and 0.1-0.4 mM, respectively. By immobilizing CD enzyme on the Nafion (R)-nsPANi composite film the maximum sensitivity of amperometric creatinine biosensor is 1300 mu A mM(-1) cm(-2).