New potentiometric and amperometric biosensors were developed for the determination of creatine. The potentiometric creatine biosensor was prepared by immobilizing urease and creatinase on poly(vinylchloride) (PVC) ammonium membrane electrode containing palmitic acid prepared by using nonactine as an ammonium-ionophore. The linear working range of the biosensor was 1.0 x 10(-5) -1.0 x 10(-3) M and the response time was about 60 s. The optimum pH, temperature, and buffer concentration were found to be 7.0, 20 degrees C, and 5 mM, respectively. The slope of the electrode was 49.2 mV/p[creatine]. The storage stabilization of the biosensor was investigated and 40-45% decrease in the response was detected after 2 months. The amperometric creatine biosensor was prepared by immobilizing creatinase (CI) and sarcosine oxidase (SO) in a poly(vinylferrocenium) matrix onto the surface of a platinum working electrode by crosslinking with glutaraldehyde (GA) and bovine serum albumine (BSA). Determination of creatine was performed by the oxidation of enzymatically generated H2O2 at +0.7 V vs. Ag/AgCl. The linear working range of the biosensor was 2.0 x 10(-5) -3.2 x 10(-4) M and the response time was about 50 s. The effects of pH, temperature, enzyme ratio and buffer concentration were investigated and optimum parameters were found to be 7.5, 37 degrees C, 2.5:1 (CI:SO) and 0.05 M, respectively. The determination of creatine in commercial creatine powder was successfully carried out with these creatine biosensors by using the standard addition and calibration curve methods. The results were in good agreement with those obtained from Jaffe method at 95% confidence level.