We present an improved method for manufacturing microfluidic structures on a polymer-based substrate, and the design of experiment (DOE) is used to extract the optimum injection parameters. The long cycle time of the injection molding causes high costs in manufacturing, and this prevents conventional techniques from being widely used for mass production. Therefore, this study adopts a new optical disk process to reduce the cycle time. The cycle time of the new method can be reduced by more than ten-fold compared with that of traditional ones. Also, this new method can prevent damage on the mirror plate of the mold. The mold system is composed of a mold insert (stamper) holder and a vacuum system to join the mold insert with the mold. In this way, the time needed to change the stamper is drastically decreased. Our proposed method has the ability to reduce the time required to insert the mold from several hours to a few minutes, to prevent damage on the mirror plate of the mold, and to decrease the cycle time of molding from several minutes to 4 sec. The DOE is applied to study the effects of molding parameters on replication rate of depth, width deviation, birefringence, tilt and surface roughness of the microfluidic substrates. The experimental results show that the proposed method is suitable for mass production.