Laser micromachining technique offers a promising alternative method for rapid production of microfluidic devices. However, the effect of process parameters on the channel geometry and quality of channels on common microfluidic substrates has not been fully understood yet. In this research, we studied the effect of laser system parameters on the microchannel characteristics of Polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), and microscope glass substrate—three most widely used materials for microchannels. We also conducted a cell adhesion experiment using normal human dermal fibroblasts on laser-machined microchannels on these substrates. A commercial CO2 laser system consisting of a 45W laser tube, circulating water loop within the laser tube and air cooling of the substrate was used for machining microchannels in PDMS, PMMA and glass. Four laser system parameters - speed, power, focal distance, and number of passes were varied to fabricate straight microchannels. The channel characteristics such as depth, width, and shape were measured using a scanning electron microscope (SEM) and a 3D profilometer. The results show that higher speed produces lower depth while higher laser power produces deeper channels regardless of the substrate materials. Unfocused laser machining produces wider but shallower channels. For the same speed and power, PDMS channels were the widest while PMMA channels were the deepest. Results also showed that the profiles of microchannels can be controlled by increasing the number of passes. With an increased number of passes, both glass and PDMS produced uniform, wider, and more circular channels; in contrast, PMMA channels were sharper at the bottom and skewed. In rapid cell adhesion experiments, PDMS and glass microchannels performed better than PMMA microchannels. This study can serve as a quick reference in material-specific laser-based microchannel fabrications.