The invention provides a high-substituted quaternary ammonium polysaccharide bioflocculant and a preparation method thereof. The preparation method comprises the following steps: alkalizing an aqueous solution of a raw material non-ionic bioflocculant in the presence of a certain amount of a catalyst at a certain water bath temperature through a wet process, slowly adding a certain amount of a dispersant in a dropwise manner, adding an etherifying agent, reacting for a period of time, precipitating, washing with water, washing with alcohol, drying, and grinding to prepare the quaternary ammonium polysaccharide bioflocculant with the substitution degree of 0.2-1.8. The method adopting the wet process has the advantages of good stability, simple operation, easy separation, high reaction conversion rate, high cost performance, and waste liquid recycling, and the quaternary ammonium polysaccharide bioflocculant has the advantages of non-toxicity, environmental protection, no secondary pollution and biodegradability, can be used for processing drinking water, printing and dyeing wastewater, coal washing water, mineral processing, oil refining wastewater, papermaking and chemical industry wastewater, and the like, and has substantial environmental and social benefits.