Display omitted •An effective film preparation method by sweep frequency pulsed ultrasound was developed.•Significant increases were observed in the mechanical properties of film by sonication.•Sonication achieved more a ordered and compact structure and a higher thermostability of film.•Ultrasonic treated film presented good antioxidant and antibacterial activities. To produce a natural food packaging film from tuna skin collagen (TSC) and chitosan (CTS) and improve its mechanical and physicochemical properties, the sweep frequency pulsed ultrasound (SFPU) was introduced as a new technology and compared with the conventional method. The optimum preparation conditions of the SFPU-TSC-CTS film were sweep frequency of 28 ± 0.5 kHz, power density of 100 W/L, sweep frequency cycle of 100 ms, pulse duty ratio of 77%, and ultrasonic time of 10 min. Significant increases in the tensile strength (27.14%) and elongation at break (16.54%) and a significant decrease in the water vapor permeability (12.15%) were observed by sonication. Thus, a moderate SFPU treatment can significantly improve the moisture resistance and mechanical properties of the film. These enhancements were achieved by a more ordered and compact structure, a good crystallinity and a higher thermostability of SFPU-TSC-CTS film, which were verified by the Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), scanning electron microscopy (SEM), X-ray diffraction (XRD), and thermal stability indexes. Moreover, SFPU-TSC-CTS film also presented good antioxidant and antibacterial activities. Therefore, SFPU was an effective auxiliary technology for improving the quality of food packaging film and can be deeply explored.