In thiswork, it has been demonstrated that the sequential inclusion of three catalytically active metals (Cu, Pt and Bi) in a glassy carbon (GC) matrix can ascribe a dual role to the trimetallic composite facilitating the oxidation and reduction of H2O2 under physiological conditions. The trimetal based nanosensing platform was constructed by sequential steps which included electrodeposition of Cu, galvanic replacement of Cu by Pt and irreversible adsorption of Bi over Pt. The electrocatalytic activity exhibited by the trimetallic composite was found to depend upon the composition and surface area. The Cu-Pt-Bi nanocomposite modified GC electrode (GC/Cu-Pt-Bi) effectively catalyzed the electrochemical oxidation and reduction of H2O2 at the potential of + 0.25 V and -0.01 V vs NCE (normal calomel electrode) respectively, with fast amperometric response, high sensitivity and low detection limit. Along with these attractive features, the modified electrode also displayed very high specificity to H2O2 with complete elimination of interference from uric acid (UA), ascorbic acid (AA), and glucose. These advantageous characteristics motivated us to build a dual mode cholesterol sensor based on H2O2 transduction by immobilizing cholesterol oxidase (ChOx) on GC/Cu-Pt-Bi. The enzyme modified nanostructured platform provided a highly sensitive and selective sensing of cholesterol coupled with a fast response time and low Km value. (C) 2017 The Electrochemical Society. All rights reserved.