This study developed a facile strategy for assembling gelatin monolayers on Titanium (Ti) surface and enabled coordinated regulation of the surface chemical composition, morphology, wetting, and charged properties. The monolayer architecture was tuned by controlling the gelatin conformation with n-alkylsulphate and n-alkylsulphonate. The increased alkyl chain length of the surfactants contributed to the decrease of Gibbs free energy and the increase of binding energy in the gelatin-surfactant, which promoted the development of a β-sheet structural framework. The β-sheet structures induced the reduction of the layer thickness, disturbed the density stacking states of nanoparticles formed from the gelatin-surfactant complex on the monolayers, and improved the adjustability of exposure density of amino groups on the thin-layer surface, resulting in a further optimization of the Ti sheets surface. The gelatin monolayers retained excellent stability even after immersion in normal saline for 7 days or in an incubator at 40 °C for 15 days. MTT assay, cell clone formation assay and cell adhesion/migration assays all demonstrated an excellent cyto-compatibility of the Ti-gelatin monolayer. This facile approach holds significant promise for applying in preparation of blood-contacting implant materials, especially cardiovascular scaffolds.