The invention discloses a preparation method of a polydopamine/titanium dioxide composite film with the controllable surface layer titanium dioxide content. The method can be used for polymer surfacemodification. The preparation method provided by the invention adopts an in-situ auto-agglutination method with controllable humidity, and comprises the steps of preparing a dopamine-Tris buffer solution and a titanium dioxide precursor sol, then soaking a polymer into the buffer solution for a certain time, taking the polymer out, pre-drying in a dampproof cabinet for a certain time, controllinga polymerization degree of dopamine on the surface, spin coating the precursor sol onto a substrate, and carrying out heat treatment. The film preparation method provided by the invention is simple, and mild in preparation environment; the film has favorable hydrophilicity and cytocompatibility, can effectively regulate the titanium dioxide content exposed on the surface of the substrate, has a certain improvement on the surface of the polymer substrate, and can be applied to the biomedical engineering fields such as cell culture in vitro and tissue engineering.