The invention relates to a formula of Lanzhou-made beef noodle soup base and a preparation method of the Lanzhou-made beef noodle soup base. The formula of the Lanzhou-made beef noodle soup base is as follows by weight ratio: 500-2500 parts of ox bones, 1000-5000 parts of beef brisket, 110-500 parts of beef tallow, 3000-10000 parts of water, 50-220 parts of salt and 14-68 parts of spices. The invention further provides the preparation method of the Lanzhou-made beef noodle soup base. The preparation method comprises the steps of precooking raw materials, carrying out high-temperature hot-pressing extraction, standing, removing residues, performing thermal flavoring reaction, blending, homogenizing, emulsifying, sterilizing, filtering, regulating soup and the like; the soup base prepared by the method has the characteristics of good fragrance, rich taste and high contents of protein and calcium, and is particularly suitable for pregnant women, the crowds in growing period and the older crowds.