Repellent coatings are critical for the development of biomedical and analytical devices to prevent nonspecific protein and cell adhesion. In this study, prevelex (polyampholytes containing phosphate and amine units) was synthesized for the fine coating of microdevices for cell culture. The dip-coating of the prevelex on hydrophobic substrates altered their surfaces to be highly hydrophilic and electrically neutral. The range of prebake temperature (50–150 °C) after dip-coating was moderate and within a preferable range to treat typical materials for cell culture such as polystyrene and polydimethylsiloxane. Scanning electron microscopy revealed a conformal and ultra-thin film coating on the micro/nano structures. When compared with poly(2-hydroxyethyl methacrylate) and poly(2-methacryloyloxyethyl phosphorylcholine), prevelex exhibited better characteristics for coating on microwell array devices, thereby facilitating the formation of spheroids with uniform diameters using various cell types. Furthermore, to examine cellular functionalities, mouse embryonic epithelial and mesenchymal cells were seeded in a prevelex-coated microwell array device. The two types of cells formed hair follicle germ-like aggregates in the device. The aggregates were then transplanted to generate de novo hair follicles in nude mice. The coating material provided a robust and fine coating approach for the preparation of non-fouling surfaces for tissue engineering and biomedical applications.