A poly (N-isopropylacrylamide) (PNIPAAm) gradient covalently anchored on a silicon substrate with a linear variation of thickness was fabricated by continuous injection of the reaction mixture (NIPAAm, CuBr and its ligand, methanol, and water) into a glass chamber containing a silicon water, whose surface had been homogeneously immobilized with bromoisobutyryl bromide (BIBB). Because of the good control of the surface-initiated atom transfer radical polymerization (SI-ATRP) technique, the thickness of the PNIPAAm brushes was linearly proportional to the polymerization time. As a result, the gradient length and sharpness could be easily controlled by the experimental parameters such as the polymerization time and the injection rate. The as-prepared PNIPAAm gradients were characterized by ellipsometry, water contact angle, and atom force microscopy to detect their alteration of the thickness, surface wettability, and morphology, confirming the gradient structure. X-ray photoelectron spectroscopy confirmed the surface composition of the PNIPAAm. In vitro culture of HepG2 cells was implemented on the gradient surfaces, revealing that the cells could adhere at 37 degrees C and could be detached at 24 degrees C when the gradient thickness was in the range of 20-45 nm. The work thus develops a method to fabricate the stable gradient surface with better quality control, and clarifies in a facile manner the appropriate thickness of the PNIPAAm brushes in terms of cell adhesion and detachment.