Poly(N-isopropylacrylamide-co-N-hydroxysuccinimide ester) was synthesized by free radical polymerization followed by gelatin grafting to obtain biocompatible thermosensitive poly(N-isopropylacrylamide- co-N-hydroxysuccinimide ester)-g-gelatin. Electrospinning was then applied to fabricate aligned thermoresponsive poly(N-isopropylacrylamide-co-N-hydroxysuccinimide ester)-g-gelatin nanofiber mats. Cell coculture study showed that this kind of nanofiber mats performed different surface adhesion to rat fibroblast cells and phoenix cells at 37 degrees C, phoenix cells can then be sorted out firstly by gradient cooling treatment. The fibroblast cells that had attached on the nanofiber mats were allowed to proliferate to reach confluence. These fibroblast cells tended to elongate along with the oriented direction of the nanofibers during culture and finally formed oriented cell sheets. This kind of aligned cell sheet could easily detach from nanofiber mats by low temperature treatment. This technique is simple and can easily harvest target cells and aligned cell sheets with minimum invasion, which has the potential to be applied in tissue engineering and regenerative medicine.