A stainless steel template for the fabrication of plastic microfluidic devices has been developed by photolithography and chemical etching technique. The preparation process of the template is simple, rapid, and low-cost. The cross sectional profiles of raised microchannels on the template are trapezoidal. The surface roughness of the templates was controlled down to 190 nm. The template can be used repeatedly to generate devices reproducibly. The microfluidic devices of poly(methyl methacrylate) (PMMA) were fabricated by in situ polymerization using the templates. The reproducibility of the fabricated microchannel is high and the relative standard deviation is 0.7% by the in situ polymerization approach. Some physical properties of the polymerized microchannels were characterized including the transparency, the thermal deformation temperature, and the dimensional information. Current monitoring was used to evaluate the electroosmotic flow within the microchannels under the electric field strength of 300 V/cm.