Abstract: Aimed to rapidly form high-quality cell sheets, poly(N-isopropylacrylamide-co-acrylic acid) (PNIPA-AA) was synthesized by free radical polymerization of N-isopropylacrylamide (NIPA) and acrylic acid (AA). The chemical structure of the synthesized PNIPA-AA was characterized by nuclear magnetic resonance spectroscopy (NMR) and Fourier infrared spectroscopy (FT-IR), and temperature sensitivity was detected by variable temperature infrared spectroscopy and ultraviolet-visible spectrophotometry. PNIPA-AA was subsequently blended with polycaprolactone (PCL) for electrospinning thermosensitive fibrous substrate of PNIPA-AA/PCL. Morphology of the electrospun fibrous PNIPA-AA/PCL substrate was observed by scanning electron microscopy (SEM) and its temperature sensitivity was confirmed through water contact angle measurement. Using mouse fibroblasts C3H/10T1/2 as model cells, effects of the fibrous PNIPA-AA/PCL substrate on the formation and detachment of cell sheets were examined. Results showed that the lower critical solution temperature (LCST) of PNIPA-AA was 33.6 ℃. PNIPA-AA possessed better electrospinnability. Electrospun fibrous substrate of the PNIPA-AA/PCL showed higher hydrophobicity at high temperature (37 ℃) and faster water infiltration rate at low temperature (20 ℃). The fibrous PNIPA-AA/PCL substrate promoted cell proliferation and extracellular matrix (ECM) secretion in C3H/10T1/2. Moreover, it took merely 10 minutes for the formed cell sheets to be completely detached by cooling down the temperature to lower than LCST, e.g., 20 ℃. And the integrity and function of the harvested cell sheets remained intact.