Among several methods to fabricate microfluidic devices, direct mechanical machining of polymers has the advantages of low cost and less lead time for fabricating new designs. In the present study mechanical micro-milling is utilized for fabricating microfluidic devices and the polymeric surfaces are investigated to understand the effect of polymer properties on surface finish during micro-milling. Here, machined surfaces of polymethylmethacrylate (PMMA) and polycarbonate (PC) materials for surface roughness and surface energy were compared with other fabrication methods. The experimental results of milling showed similar average roughness (Ra) on PMMA and PC materials but the weissienberg effect (climbing effect of polymers) was observed in the machined area. For further quantitative analysis of surface integrity, surface energy on machined surfaces was performed using Fowke’s theory. Surface energy measured was compared with other surfaces obtained from various fabrication methods like hot embossing and lithography. 3D surface topography, surface roughness, and burr formation were visualized using contact and non-contact profilometers and a Stereomicroscope. Results indicate that the machining of PMMA is the preferable choice for fabricating microfluidic devices compared to PC. Furthermore, microfluidic channels with serpentine channels are machined with a depth of about 50µm and a width of 200µm. The functioning of these devices is evaluated for inertial focusing phenomenon and blood plasma separation achieving flow rates of 0.1 to 0.5ml/min. This study with quantitative measurements validates the choice of material for fabricating microfluidic devices and provides insight into the effects of the influence of surface characteristics while machining polymers.