Chicken feet, aplenty by-products in the chicken industry, are rich in collagen and contain abundant amino acids so that it can be used as an important source for the collagen market. Pepsin-soluble collagen (PSC) was extracted from chicken leg skin and explored the effects of single- and tri-frequency ultrasound on the self-assembly and vitro digestion characteristics. By the diverging and tri-frequency ultrasound reactor, PSC was treated with 20 kHz/270w (C20H5m), 40 kHz/270w (C40H5m), 60 kHz/270w (C60H5m), 20/40/60 kHz/90w x 3 (CtH5m) for 5 min. Results showed that ultrasound could accelerate the process of collagen self-assembly, and 60 kHz/270w was the fastest. Microfiber diameters of C60H5m were 65-89 nm, which was significantly lower than the control without ultrasound (80-161 nm). The digestion results indicated polypeptides with relative molecular weights founded in the range 200-5000 Da were exceeded 85%. The final digested product had the highest content of oligopeptide, consistent rheological properties, and elastic behavior. The cavitation and mechanical of ultrasound have effects on the self-assembly process and collagen gel structure and digestion characteristics, which is of great significance for the development of the chicken industry and collagen market.