Several studies have demonstrated the ability of laser for micro-channels fabrication. There are some important aspects that must be considered to use laser for machining process, such as machining method, type of laser and interaction between laser and workpiece. In this research, CO2 laser machine was used as a tool to fabricate a micro-channes on acrylic. Some parameters that can influence the outcome of the cuts were setup, which are focus distance on Z axis, laser power, cutting speed, and the repetition of cutting process (number of pass). Cutting results were width and depth of cut. Observation was made by using digital microscope as measuring tool, and measurement result was analyzed by using Anova method. The results of the analysis shows that the parameter of laser power has a dominant influence on depth of cut, and then parameter of cutting speed and number of pass. By determining width and depth of the cut based on given parameters, it will be easier to take a shape and size on micro-channel fabrication.