Abstract: In order to improve the utilization rate of bovine bone resources, fresh bovine bone was used as raw material, four kinds of bovine bone collagen (ASC1, ASC2, PSC1 and PSC2) were extracted by acetic acid, citric acid, acetic acid and pepsin, citric acid and pepsin, the physicochemical properties and structural characteristics were studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), surface hydrophobicity and other indicators. The results showed that PSC1 had the highest yield, which was 3.23%, the maximum ultraviolet absorption peak of the collagen extracted by four kinds of extraction methods were found around 230 nm. SDS-PAGE and infrared spectroscopy analysis showed that four kinds of collagen were composed of α₁, α₂ and β subunits, which belonged to type I collagen and the space structure of bovine collagen was preserved intact. Scanning electron microscopy showed that the fiber network structure of the four kinds of collagen (acid method and enzyme method) were relatively intact, among the network structure of the collagen extracted by acid method (ASC1 and ASC2) was evenly distributed. The collagen extracted by enzymatic method (PSC1 and PSC2) had obvious fibrosis. There was no significant difference in the physicochemical properties of the four kinds of collagen, but there were some differences in the microstructure. This study would provide a theoretical basis for improving the development of high value application and deep processing technology of livestock and poultry by-products.