Tresyl chloride (2,2,2-trifluoroethanesulfonyl chloride)-activated method is an easy and simple method for immobilizing biological molecules onto a titanium surface. Tresyl chloride was directly dropped onto a titanium surface without any solvent. Tresylated titanium disks were immersed in a protein or peptide solution to give protein- or peptide-immobilized titanium. Frontier molecular orbital calculations suggested that tresylation of the OH compound improved its reactivity towards the amine groups of protein compounds. Quartz-crystal microbalance-dissipation measurements suggested that ionic interaction between the oxygen of the terminal hydroxyl groups of titanium and the nitrogen of fibronectin was important for fibronectin immobilization. Initial cell attachment was enhanced by fibronectin or collagen immobilization. Fibronectin and fibronectin-derived peptide immobilization improved the expression of specific genes related to bone formation. The immobilization of cytokine onto titanium enhanced bone formation following its implantation into rat femur defects. The tresyl chloride-activated method is useful for immobilizing biological molecules onto titanium surfaces.