The assembly of multilayer films of gelatin onto glass substrates using layer-by-layer and lift-off (LbL-LO) technology to modify the surface topography and chemistry properties of in vitro cell culture scaffolds is described. The ability to generate such nanoscale systems containing cell-adhesive materials on optically transparent substrates with microscale lateral dimensions, nanoscale vertical dimensions, molecular vertical precision, and flexibility in material selection has important implications for tissue engineering, drug discovery, and basic research in cell biology. Toward this goal, a systematic study on the electrostatic adsorption properties of fluorescein 5-isothiocyanate-gelatin B (FITC-gelatin) was completed. In addition, the integration of protein nanoassembly with microlithographic feature definition was used to pattern three-dimensional FITC-gelatin nanofilms on planar glass substrates. The experimental results indicate that FITC-gelatin is negatively charged at pH 9 and can be alternately assembled with a positively charged polyion, poly(diallyldimethylammonium chloride) (PDDA), to form multilayer films on solid templates with thickness of 5-10 nm per bilayer. Furthermore, images of protein/polymer nanocomposites indicate that LbL-LO is an efficient way to realize the designed substrates. These findings will benefit future research on cell culture and tissue engineering that require methods of generating protein patterns to fabricate novel in vitro cell culture systems.