The invention relates to a preparation method of a polyphenol biomass flocculating agent, and belongs to the field of fine chemicals. The preparation method comprises following steps: firstly acrylamide is uniformly mixed with water, potassium peroxodisulfate is added, and an obtained mixture is stirred at 80 DEG C for 2h of reaction; an obtained product is filtered and washed, and an obtained filter residue is dried at 85 to 90 DEG C until weight is constant so as to obtain a modified polyphenol biomass; the modified polyphenol biomass particles, potassium peroxodisulfate, and methacryloyloxyethyl trimethyl ammonium chloride are added into acrylamide step by step, reaction is carried out for a certain period of time, and an obtained product is dried and smashed so as to obtain the polyphenol biomass flocculating agent. The polyphenol biomass flocculating agent can be used for treating domestic wastewater, and waste water of the fields such as food, paper and paper pulp, light industry, chemical industry, and oil and gas processing, is nontoxic, is friendly to the environment, possesses no potential safety hazard, and is low in cost. The raw materials of the polyphenol biomass flocculating agent are cheap, and are widely available; flocculation effect is excellent; and the polyphenol biomass flocculating agent is convenient to degrade, and is safe to use.