Poly(ethylene terephthalate) (PET) has a low glass transition temperature. Therefore it has significant applications in fields where low bonding temperature is needed. But PET microfluidic chip production using a hot embossing/bonding method has rarely been reported. In this study, hot embossing was conducted for a micro-feature's fabrication on a PET substrate, and a special temperature–pressure profile was used to achieve high replication accuracy without a vacuum; plasma surface treatment was used to improve the bonding capability of PET material, a cover plate was bonded with a substrate at a low temperature of around 63 °C, and VB2 was used as a separation solvent to test the capability of the PET microfluidic chip. The results show that high replication accuracy can be achieved using the new hot embossing process without a vacuum. Plasma surface treatment has increased the surface energy of the PET substrate and hot bonding can be achieved in low temperature. Plasma treatment has also changed the hydrophobic property of PET material; electrophoresis has been conducted successfully.