CO2 laser direct-writing ablation micromachining technique is used to fabricate the microchannel on the polymethyl methacrylate (PMMA) substrate. The correlation between the process parameters (the beam translational velocity and the processing number of times) and the micromachining quality (the width and the depth of the microchannel) is investigated. For a given processing times, the depth and width of microchannel both decrease along with the enhancement of beam translational velocity. At fixed beam translational velocity, the depth and width are direct proportion to the processing times, respectively. The microchannel is fabricated on different PMMA substrates with smooth surface, rough surface and surface adhering water. The micromachining quality and the surface roughness of the microchannel are compared. The microchannel with a hydraulic diameter of 80 μm is fabricated under two experimental conditions, and the comparative experiment has been performed on the microchannel with the relative roughness. It is found that the relative roughness is minished effectively when a laser is used to make ablation of the microchannel on the PMMA substrate with surface adhering water. The improved fabrication procedure will be used to increase the availability of CO2 laser direct-writing ablation technique on the micromachining of the PMMA microfluidic chip.