CO2 laser machining technology is a wide and low-cost method for fabrication of microfluidic chips on polyethylene terephthalate (PET). In this paper, the influence of CO2 laser parameters including laser power and laser moving velocity on the depth and width of PET microchannel are studied. Laser power is set from 4 W to 20 W and laser moving velocity is set from 5 mm/s to 25 mm/s in the experiment. Compared with experimental results, some rules for the depth and width on laser parameters are obtained. The depth and width of the microchannel increase with the increase of laser power at the same laser moving velocity. However, the depth and width of the microchannel first increase and then decrease with the increase of laser moving velocity at the same laser power. The PET microfluidic chip is fabricated by a hot bonding machine.