A method to fabricate biocompatible polymer microfluidic systems with integrated electrical and fluid functionality has been established. The process flow utilizes laser ablation, microstenciling, and heat staking as the techniques to realize multi-layered polyimide based microsystems with microchannels, thru and embedded fluid / electrical vias, and metallic electrodes and contact pads. As an application of the fabrication technology, a six layer multi-functional cellular analysis system has been demonstrated. The electrophysiological analysis system contains fluid microchannel / via networks for cell positioning and chemical delivery as well as electrical detectors and electrodes for impedance spectroscopy and patch clamping studies. Multiple layers of 50.8µm thick Kapton® sheets with double-sided polyimide adhesive layers were used as the primary material-of-construction. Microchannels with widths of 400µm as well as thru hole vias with 3.71µm diameters (aspect ratios of over 12:1) were laser ablated through the polyimide sheets using an excimer laser and a CO2 laser. Electrical traces and contact pads with features down to 20µm were defined on the flexible polyimide sheets using microstenciling. The patterned layers were bonded using heat staking at a temperature of 350°C, a pressure of 1.65MPa for 60 minutes. This multi-layer technology can be used to create microfluidic devices for many application areas requiring biocompatibility, relatively high temperature operation, or a flexible substrate material.