Abstract: The present disclosure belongs to the technical field of biochemical engineering, and relates to a preparation method of ?-galactosidase. The preparation method includes the following steps: step 1, seeding Bacillus circulans into a Luria-Bertani (LB) medium for culture and activation and then into a seed tank for culture to obtain a seed culture broth; step 2, introducing the seed culture broth into a fermentor containing a fermentation medium for fermentation to obtain a fermentation broth of B. circulans; and step 3, filtering or centrifuging the fermentation broth of B. circulans to remove cells to obtain a ?-galactosidase liquid; the fermentation medium includes lactose, galactose, phytone, corn meal, yeast extract, a phosphate salt, a carbonate salt, and water. According to the method, fermentation time is short, production period is shortened, and specific activity of the resulting enzyme liquid is high.