The ability to fabricate high-quality colloidal photonic crystal (CPC) films in large areas is critical in many applications, ranging from flexible displays, security devices to optical enhancement. Herein, a large-scaled CPC film with crack-free structure and uniform optical performance is prepared via a hydrogen-bond-assisted method. The crack-free CPC film is ascribed to the intermolecular hydrogen bonds between carboxyl (COOH) moieties of poly(styrene-methyl methacrylate acrylic acid) microspheres and isocyanate (NCO) moieties of polyurethane. Furthermore, the as-prepared CPC film is applied as a Bragg reflection mirror for fluorescence enhancement. It is demonstrated that a fourfold enhancement of fluorescence signal is achieved when the stopband of CPCs overlaps with the emission wavelength of quantum dots. This simple method of preparing large-area and crack-free CPC film is promising for developing efficient optical devices.