We investigated the electrochemical detection of aspartate transaminase (AST) and alanine transaminase (ALT) by using a multienzyme-modified electrode surface. Determination of the activities of transaminases in human serum is clinically significant because their concentrations and ratios indicate the presence of hepatic diseases or myocardial dysfunction. For electrochemical detection of AST and ALT, enzymes that participate in the reaction mechanism of AST and ALT, such as pyruvate oxidase (POX) and oxaloacetate decarboxylase, were immobilized on an electrode surface by using an amine-reactive self-assembled monolayer and a homobifunctional cross-linker. In the presence of suitable substrates such as L-aspartate (L-alanine) and α-ketoglutarate, AST and ALT generate pyruvate as an enzymatic end product. To determine the activities of AST and ALT, electroanalyses of pyruvate were conducted using a POX and ferrocenemethanol electron shuttle. Anodically generated oxidative currents from multienzyme-mediated reactions were correlated to AST and ALT levels in human plasma. On the basis of the electrochemical analysis, we obtained calibration results for AST and ALT concentrations from 7.5 to 720 units/L in human plasma-based samples, covering the required clinical detection range.