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 laser power and the beam translational velocity) and the micromachining quality (the depth and the width of the microchannel) is investigated. A laser power of 4.5 W and beam translational velocity of 76.2 mm/s is used to make ablation of the microchannel with a hydraulic diameter of 100 μm on the PMMA substrate. The time for the ablation process is 30 s. This technique is steady and efficient in the batch production. The microchannel has the same Raman spectra as that of the PMMA substrate. The spectra shows that they have the same chemical effect on the polymerase chain reaction (PCR) mixture. The presence of the bumps of resolidified material on the edges of the microchannel has no influence on the hot-press bonding result and the PCR microfluidic chip could satisfy the pressure and the sealing requirement in PCR process. An amplification of a DNA template with 180 bp fragment of arabidopsis thaliana is successfully performed with this chip, in less than 30 min. This result exhibits the availability of CO2 laser direct-writing ablation technique on the micromachining of the PMMA continuous-flow PCR microfluidic chip.