In the last two decades, there have been significant advancements in the development of more physiologically relevant organ-on-a-chip (OOC) systems that can mimic the tissue microenvironment. Despite the advantages of these microphysiological systems, such as portability, the ability to mimic physiological flow conditions, and the reduction of reagents required for preparation and detection, they lack real-time detection of analytes with high accuracy. To address this, biosensor technologies have been integrated with OOC systems to enable simultaneous analysis of different analytes in a single device. However, integrating biosensors with OOC systems is challenging due to the competing demands for low-cost, simple fabrication processes, and speed. This study presents the fabrication of a glucose sensing device integrated with a liver-on-a-chip (LOC) platform. The conductive PLA-based three-electrode system was printed using FDM 3D printing technology to simplify the fabrication process. The sensitivity of the glucose biosensing device was enhanced by adding multi-walled carbon nanotubes on the electrodes. The biosensing integration study using a perfusion-based LOC showed the stability, biocompatibility, and sensitivity of the glucose sensing devices. Furthermore, drug toxicity studies on the LOC platform demonstrated the device’s ability to detect a broad range of glucose concentrations and its enhanced sensitivity.