Polydimethylsiloxane (PDMS)-based microfluidic chips are extensively utilized across diverse disciplines. Traditionally, photolithography is used for mold making, but it’s expensive, time-consuming, limited in microchannel design, and cannot generate a wide range of varied cross-sections for the microchannel. This research introduces micro-milling, a more straightforward and more versatile approach, to create precise Poly(methyl methacrylate) (PMMA) molds. This method ensures the final chip’s accurate size, shape, and surface roughness. First, the surface roughness of PMMA is optimized based on the input parameters, which include spindle speed, feed rate, depth of cut, and stepover. The experiment showed that the minimum surface roughness was achieved at 0.2 μm with the following parameters: cutting speed of 20,000 rpm, depth of cut of 10 μm, feed rate of 300 mm/min, and step over of 30%. Next, the optimized parameters for fabricating PMMA micromolds were applied to fabricate PDMS microchannels. The mold’s surface roughness has been optimized to below 0.2 μm; the smallest width of the microchannel was measured at 50 μm.