This study used Langmuir-Schaefer (LS) method to produce thermo-responsive poly(N-isopropylacrylamide) (PIPAAm) modified surface. Dodecyl terminated-PIPAAm (PIPAAm-C12) was synthesized by reversible addition-fragmentation chain transfer radical polymerization. PIPAAm-C12 was dropped on an air-water interface and formed Langmuir film by compressing. A surface pressure measurement revealed that PIPAAm-C12 was floated and Langmuir films were formed on the interface. And the Langmuir film was transferred on a hydrophobic substrate to produce PIPAAm-C12 transferred surface (PIPAAm-LS surface). In the results of atomic force microscope, attenuated total reflection Fourier transform infrared spectroscope, and X-ray photoelectron spectroscope measurement, the transference of Langmuir films was demonstrated and densities could be precisely controlled. Ellipsometric measurements of PIPAAm-LS surfaces showed that the thicknesses of the surfaces were less than 10 nm. Cell adhesion and detachment were observed on the PIPAAm-LS surfaces. The amount of adhered cells on all LS surfaces was found to be similar on the control hydrophobic substrate at 37 °C. In regard to cell detachment, adhering cells rapidly detached themselves with higher densities and shorter PIPAAm-C12 molecules. In this method, the effect of densities and molecular weights on cell adhesion and detachment were observed. Our method should be proved novel insights for investigating cell adhesion and detachment on thermo-responsive surfaces.