Assembly of colloidal arrays has attracted much attention over the past decade. Surface property and topology play important roles in the assembly of colloidal patterns and layers on various substrates. In this paper, we report on a novel micropattern-assisted nanoassembly (μPAN) method to organize highly ordered nanocolloidal arrays onto a nonfouling polymer surface. Polyethylene glycol (PEG) hydrogel is used as the substrate material in the study, for its desired biological properties, low surface energy and photopatternability. A highdensity array of PEG microwells is first fabricated on glass substrates with the minimal feature resolution of 1 Jim using photolithography method. By simply controlling the colloidal concentration of the dipping solution, the dimensions of the microwells and pulling speed of the substrate, various well organized nanocolloidal patterns are assembled inside the PEG microwells. In addition, the effect of surface property is experimentally investigated. After oxygen plasma treatment, the nonfouling property of the PEG surface deteriorates significantly, leading to a complete surface coverage of nanocolloidal beads. The novel μPAN method allows organizing highly ordered nanocolloidal arrays in a predictable and robust fashion, and has potential applications in photonic crystal fabrication, biological sensing, analytical detection and nanoassembly.