Pentacyanoferrate-bound poly(1-vinylimidazole) (PVI[Fe(CN)(5)]) was selected as a mediator for ampermetric creatinine determination based on the reductive H2O2 detection. Creatinine amidohydrolase (CNH), creatine amidohydrolase (CRH), sarcosine oxidase (SOD), peroxidase (POD), and PVI[Fe(CN)5] were crosslinked with poly(ethylene glycol) diglycidyl ether (PEGDGE) on a glassy carbon (GC) electrode for a creatinine biosensor fabrication. Reduction current was monitored at -0.1 V in the presence of creatinine and O-2. It is revealed that PVI[Fe(CN)(5)] is suitable as a mediator for a bioelectrocatalytic reaction of POD, since PVI[Fe(CN())5] neither reacts with reactants nor works as an electron acceptor of SOD. The amounts of PVI[Fe(CN)(5)], PEGDGE, and enzymes were optimized toward creatinine detection. Nafion as a protecting film successfully prevented the enzyme layer from interferences. The detection limit and linear range in creatinine determination were 12 mu M and 12-500 mu M (R-2 = 0.993), respectively, and the sensitivity was 11 mA cm(-2) M-1, which is applicable for urine creatinine tests. The results of the creatinine determination for four urine samples measured with this proposed method were compared with Jaffe method, and a good correlation was obtained between the results. (C) 2013 Elsevier B.V. All rights reserved.