This study develops an improved method for generating aluminum mold inserts used in the replication of polymer-based microfluidic chip. Since molding masters that are suitable for microfluidic chip replication must have features whose dimensions are of the order of tens to hundreds of microns, micro electrical discharge machining is employed herein to fabricate an aluminum mold insert of a microfluidic chip. The width and depth of the aluminum mold insert for the microfluidic chip are 61.50 μm and 49.61 μm, respectively. The surface roughness values of the microchannel and the sample reservoir in aluminum mold insert for the microfluidic chip are 59.92 nm and 34.34 nm, respectively. PMMA material is adopted as the molded microfluidic chip that is produced by micro-hot embossing molding. The PMMA material can replicate the microchannel and sample reservoir very well when the aluminum mold insert is used in micro-hot embossing molding. The results indicate that the most important parameter in the replication of molded microfluidic chip is the embossing pressure, which is also the most important parameter in determining the surface roughness of the molded microfluidic chip.