Phage-displayed titanium (Ti) binding-peptides (TBPs) have potential applications in immobilizing functional proteins on the solid substrates. Ti/TiO2 particles or films growing in the Si or SiO2 surfaces have been used for biopanning Ti binding-peptides. Besides, 2D aligned films prepared from engineered M13 bacteriophages (phage) are being studied for applications in tissue-regeneration and energy devices. In this study, peptides with specific affinity to acid-alkali treated Ti surface were biopanned by phage display technology and identified by specific fluorescent stain and titration measurement. The average n mbers of hydroxyl-containing residues, aspartic acid and histidine residues in the identified 8 new unique peptides are distinctly higher than those in the overall peptide-bearing phage library. The TBP54-phage shows higher binding affinity to the substrate than the others. The Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) characterizations indicate that the coordination and electrostatic interactions together control the specific binding of these phages to the Ti surface treated with acid-alkali. To obtain a uniform film, the TBP54-M13 phages were treated by polyvinyl pyrrolidone and then coated onto the pretreated Ti surface and acid-alkali treated Ti surface. The resultant film on acid-alkali treated Ti surface exhibits uniform nanostructure, favorable stability and substrate selectivity. The phage film could be used for surface multi–functionalization and patterning of Ti implants further.