A micro-ablation of polymethyl methacrylate (PMMA) is carried out by CO2 laser. The effect of the process parameters on the depth and width of the channel is further studied. The rule of the process parameters how to affect the groove depth and width is obtained by the experimental analysis. The results show that the laser line energy density has an exponential relation with the channel width. Through the experiment, we achieve the experiental formula about the line energy density and groove width and the decisive coefficient R2 is 0.96. When the line energy density is less than 300 J/m, the depth of ablated channel has a linearity relation with the line energy density, but this relation is deviated for the line energy density over 300 J/m. By the law of conservation of energy, a model about the channel depth and line energy density is set up. At the situation of low line energy density less than 300 J/m, the calculated results agree well with the experiment. At last, with optimization of process parameters, we make a fine biochip with width of 170 μm and depth of 180 μm.