In this paper, we report on the effect of patterned non-adhesive hydrogel nanosurfaces on the self-assembly of highly ordered colloids. Polyethylene glycol (PEG) hydrogel is employed as the substrate material in the study, for its desired non-adhesive property, and biocompatibility as well as photopatternability. Ultrafine PEG features are photopatterned onto glass substrates with minimal feature resolution of 500 nm using ultraviolet or deep ultraviolet exposure. By simply controlling the colloidal concentration of the nanoassembly solutions and the dimensions of the wells, a range of highly organized nanocolloidal patterns are formed inside the PEG wells. Unlike the traditional surface modification techniques, ours takes advantage of the unique non-adhesive property of PEG hydrogels to achieve extremely high selectivity in the pattern-assisted nanoassembly. Our experiments show that with oxygen plasma treatment, the non-adhesive property of the PEG surface deteriorates significantly, leading to non-selective assembly with complete surface coverage of nanocolloidal beads under the same processing condition. Therefore, benefiting from the unique non-adhesive surface property, the pattern-assisted nanoassembly method enables a highly predictable and robust process for colloidal nanofabrication, and the obtained nanocolloidal arrays with well organized patterns could potentially find applications in photonic crystal fabrication, biological sensing and analytical detection.