The invention provides a preparation method of an anionic glucan flocculating agent. The method comprises the following steps: 1, preparing a sucrose solution by adopting an acetic acid-sodium acetate buffering solution with the pH value of 5.0, adding dextransucrase to the sucrose solution, carrying out an enzymatic catalytic reaction, terminating the enzymatic catalytic reaction when the dynamic viscosity of the reaction solution reaches 8000-10000CPS in order to precipitate polymeric glucan, washing, and dissolving the washed polymeric glucan in water to obtain a polymeric glucan solution with the solid content of 5-10%; and 2, adding potassium persulfate and sodium bisulfite to the polymeric glucan solution as initiators, maintaining the temperature in a range of 50-70DEG C for 15-20min, adding a sodium acrylate solution as an anion monomer, carrying out a grafting copolymerization reaction, terminating the grafting copolymerization reaction, adopting ethanol to precipitate a crude grafted product, washing the crude grafted product by using ethanol, and re-dissolving the washed product in water to prepare the milky viscous liquid anionic glucan flocculating agent.