The invention provides a method of extracting myoglobin from beef. The method includes the steps of adding a Tris-HCl mixed buffer solution to a beef meat sample, homogenizing, standing, centrifuging,adding (NH4)2SO4 and standing, centrifuging, adding a Tris-HCl buffer solution b, performing dialysis, and eluting with a NaCl mixed solution in a chromatography column to obtain the myoglobin in thebeef. According to the method, the pH value of the Tris-HCl mixed buffer solution, the extraction time and the saturation of the myoglobin salt precipitation solution are optimized to increase the extraction amount of myoglobin; the Tris-HCl mixed buffer solution contains glycerin and dithiothreitol, the glycerin maintains the structural stability of the myoglobin and facilitates rapid precipitation, and the dithiothreitol maintains the reductive and biological activity of the myoglobin; the solid (NH4)2SO4 is added for fractional salt precipitation to increase the purity of the myoglobin, the temperature is kept at 4 DEG C to prevent degeneration and oxidation of the myoglobin, residual salt ions and other small molecular proteins can be effectively removed through the dialysis to improve the purity of the myoglobin, and the separation by gel filtration chromatography carried out at last does not destroy the structure and function of the myoglobin.