A biodegradable, temperature-sensitive dextran-allyl isocyanate-ethylamine (TSDAIE) as a nonenzymatic cell detachment polymeric substrate for human endothelial progenitor cells (EPCs) is developed and examined. The lower critical solution temperature of TSDAIE is determined; its phase transition occurrs at 18 to 22 °C. For EPC culture, cell culture flasks are coated with TSDAIE and type I collagen. The TSDAIE coating enables EPC detachment when the culture is cooled to 4 °C. The concentration of TSDAIE affects EPC attachment, which is thereby used to optimize the concentration of TSDAIE for coating. At the determined optimal concentration, TSDAIE is found to be compatible for use in EPC culture as revealed by cell attachment, spreading, proliferation, and phenotype. Overall, biodegradable TSDAIE shows promise for applications that culture and expand EPCs including vascular regenerative medicine and tissue engineering.