As an alternative to SU-8 soft lithography, a new silicon mold process of fabricating PDMS microchannel chips was proposed. A picosecond laser is used to cut through a 550 μm thick silicon wafer and generate the original microchannel pattern with a 50 μm minimum feature size. This single-crystal silicon pattern, with the edge debris caused by laser cutting being trimmed off by a KOH solution and with the protection field oxide layer being removed by BOE afterwards, firmly resided on a glass substrate through the anodic bonding technique. Four-inch wafers with microchannel patterns as the PDMS mold cores were successfully bonded on Pyrex 7740 or Eagle XG glass substrates for the follow-up PDMS molding/demolding process. This new maskless process does not need a photolithography facility, but the laser cutting service must be provided by professional off-campus companies. One PDMS microchannel chip for particle separation was shown as an example of what can be achieved when using this new process.