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The paper demonstrates four different polymer substrate microchannels are fabricated by CO2 laser machine. The four different polymer substrates are Polymethyl-methacrylate (PMMA), Polycarbonate (PC), Polystyrene (PS) and Polyethylene Terephthalate (PET), respectively. A number of microchannels are obtained and all roughness is measured. The four different polymer substrate microchannels are processed with different processing parameters. Laser power is set from 4W to 32W and laser cutting speed is set from 5mm/s to 30mm/s. The results show the roughness of PS substrate microchannel is lower than that of other three polymer substrate microchannels at the same parameters. When laser power is below 4W, the roughness of four polymer substrates are similar. The roughness of different polymer substrate microchannels decreases with the increase of laser power. The roughness of different polymer substrate microchannels also happens to change with increase of laser cutting speed.