For efficient therapeutic use of human mesenchymal stem cells (hMSCs), maximizing their self-renewal performance and multipotency must be fully retained. However, conventional trypsin-based cell passaging methods are known to damage the attached cells to be detached because of the inherent corrosive nature of trypsin, and continuous passaging substantially degrades the self-renewal and differentiation capacity of hMSCs. Therefore, it is imperative to secure a damage-free passaging method that supports cell growth as well as their stem cell function. Here, an enzyme-free cell detachment method using a poly(ethylene glycol dimethacrylate) (pEGDMA)-coated surface is developed, which allows for reduced integrin-dependent cell adhesion. Cell detachment can be facilitated simply by treating the plated cells on the pEGDMA surface with Ca2+ and Mg2+-depleted DPBS. Spontaneous cell detachment occurs within 10 min with the full retention of the cell viability and proliferation ability of hMSCs. Especially, the detachment method can minimize the surface protein damage of hMSCs compared to the conventional trypsin treatment and preserve the self-renewal property and differentiation capacity even with an increased passage number over 10. The developed enzyme-free detachment method using the pEGDMA-coated surface is highly promising for a culture platform to broaden its application to the field of tissue engineering and regenerative medicine.