The planar-type amperometric creatinine biosensor employing an additional enzyme layer containing creatine kinase and adenosine triphosphate was developed to eliminate severe interference from creatine. In the additional enzyme layer, an interfering substance, creatine is converted to noninterfering product, phosphocreatine. Furthermore, the carbon electrode electroplated with Pt black(Pt-B) was employed to fabricate creatinine biosensors with improved sensor performance(e.g., sensitivity, reliability, and reproducibility). The creatinine levels in an unknown sample were determined within less than 5% errors using creatinine microsensors equipped in a flow-cell cartridge.