Creatine bienzymatic potentiometric biosensors were prepared by immobilizing urease and creatinase with four different procedures on poly(vinylchloride) (PVC) ammonium membrane electrode containing palmitic acid prepared by using nonactine as an ammonium-ionophore. The analytical characteristics were investigated and were compared with those of the biosensor prepared by using carboxylated PVC. The effects of pH, buffer concentration, temperature, enzyme concentration and stirring rate, and enzyme immobilization procedures on the response to creatine of the biosensor were investigated. The linear working range and sensitivity of the biosensor were also determined. The creatine biosensor prepared by using the PVC membranes containing palmitic acid showed more effective performance than those of the carboxylated PVC based biosensors. Additionally, creatine assay in serum was successfully carried out by using the standard addition method without any pretreatment. The results were good agreement with those obtained from Jaffe methods at 95% confidence level. (c) 2006 Elsevier Ltd. All rights reserved.