This manuscript describes a simple and versatile approach to engineering surface roughness gradients via combination of microfluidics and photo-polymerization. Through UV-mediated polymerization, N-isopropylacrylamide with concentration gradients are successfully grafted onto PDMS surface, leading to diverse roughness degrees on the obtained PDMS substrate. Furthermore, the extent of surface roughness can be controllably regulated via tuning the flow rate ratio between the monomer solution and deionized water. Average roughness ranging from 8.050 nm to 151.68 nm has well been achieved in this work. Such PDMS samples are also demonstrated to be capable of working as supporting substrates for controlling cell adhesion or detachment. Due to the different degrees of surface roughness on a single substrate, our method provides an effective approach for designing advanced surafecs for cell culture. Finally, the thermosensitive property of N-isopropylacrylamide makes our sample furnish as another means for controlling the cell detachment from the substrates with correspondence to the surrounding temperature.