Thready stripe-patterned thermo-responsive surfaces were prepared and their surface properties were characterized. Prepared 3 μm wide stripe-patterned surfaces were evaluated by observing the adhesions and detachments of three types of cells: HeLa cells (HeLas), human umbilical vein endothelial cells (HUVECs), and NIH-3T3 cells (3T3s). Although cell adhesion and detachment in response to temperature were observed on all cells on a conventional thermo-responsive surface without patterns, the thermo-responsive surface with a 3 μm striped-pattern exhibited various cell adhesion properties. HeLas hardly adhered to the patterned surface even at 37 °C. On the other hand, although HUVECs adhered on the patterned surface at 12 h after incubation at 37 °C, the adhered HUVECs detached themselves after another 12 h incubation at 37 °C. 3T3s adhered to the patterned surface at 37 °C and detached themselves after reducing temperature to 20 °C. A mixture of HeLa, HUVEC and 3T3 was separated using their different specific cell-adhesion properties, and the composition of cells was analyzed by a flow-cytometry. As a result, the conventional thermo-responsive surface with a stripe-pattern was found to function as a cell-separating interface by using specific cell adhesion properties.