On-line separations-based sensors employing microdialysis (MD) coupled to microchip electrophoresis (ME) enable the continuous monitoring of multiple analytes simultaneously. Electrochemical detection (EC) is especially amenable to on-animal systems employing MD-ME due to its ease of miniaturization. However, one of the difficulties in fabricating MD-ME-EC systems is incorporating carbon working electrodes into the device. In this paper, a novel fabrication procedure is described for the production of a PDMS/glass hybrid device that is capable of integrating hydrodynamic MD flow with ME-EC using a flow-gated interface and a pyrolyzed photoresist film carbon electrode. This fabrication method enables the reuse of carbon electrodes on a glass substrate, while still maintaining a good seal between the PDMS and glass to allow for pressure-driven MD flow. The on-line MD-ME-EC device was characterized in vitro and in vivo for monitoring analytes in the dopamine metabolic pathway. The ultimate goal is to use this device and associated instrumentation to perform on-animal, near-real time in vivo monitoring of catecholamines.