The invention relates to a hepatocyte growth-promoting factor. A preparation method for the hepatocyte growth-promoting factor comprises the following steps of: selecting materials; treating tissues; mixing the materials and injection water at room temperature, uniformly stirring, and freezing homogenate liquid to the temperature of between -20 and -35 DEG C; unfreezing the homogenate liquid which is subjected to freezing storage for at least seven days, then heating to the temperature of between 83 and 85 DEG C, immediately removing a heating source, filtering, and adjusting the pH value to be 6.5; filtering by using a hollow fiber column with the molecular weight cutoff of 20,000, then filtering by using an ultrafiltration membrane with the molecular weight cutoff of 10,000, freezing ultrafiltrate to the temperature of between -20 and -30 DEG C, and storing in a sealing way; and unfreezing the ultrafiltrate which is subjected to freezing storage for at least seven days, filtering, concentrating, filtering the concentrated solution by using a hollow fiber column with the molecular weight cutoff of 10,000, degerming by using a filtration membrane with the aperture of 0.22 micrometer, performing split charging, and storing at the temperature of below -18 DEG C. The invention also relates to a preparation and application of the hepatocyte growth-promoting factor. The hepatocyte growth-promoting factor has high activity and high yield and is safely used.