The development of new sensing platforms for the rapid, real-time, and onsite detection of metabolic compounds could provide significant opportunities for medical science, and healthcare applications. Microplates are standard tools that are widely used in cell biology, tissue engineering, or medical diagnosis. Here, we demonstrate for the first time the microplate-integrated amperometric biosensors for the rapid and sensitive detection of glucose and lactate. The sensor is constructed by printing carbon-graphite mediator paste as the working electrode, and Ag/AgCl paste as both reference and counter electrodes. The working electrode is then immobilized with glucose oxidase or lactate oxidase. The sensor shows a sensitive and rapid detection of glucose or lactate with a detection range from micromolar to millimolar and a short detection time of about 1 min. It is expected that the methodology can provide rapid, real-time, and onsite platforms for detecting a variety of metabolic compounds, and could provide new avenues for the fundamental studies and practical applications in cell biology, tissue engineering, or medical diagnosis. (C) 2019 The Electrochemical Society.