The invention discloses a preparation method of a fresh water composite microorganism bottom modifier. The preparation method comprises the steps of preparation of a fermentation culture medium, fermentation, adsorption and crosslinking. The method comprises the following specific steps: firstly, mixing bran powder, corncob powder, starch wastewater and a fermentation synergist to obtain the fermentation culture medium; introducing a mixed bacterial solution of rhodococcus erythropolis, saccharomyces cerevisiae and bacillus subtitles for fermentation culture, and purifying to obtain a microbial flocculant; then, adsorbing sesame straw activated carbon, enzymatic liquid, the microbial flocculant and a microbial preparation; performing crosslinking with a crosslinking agent to obtain the fresh water composite microorganism base modifier. The preparation method has the beneficial effects of easiness and feasibility, higher yield and productive rate of polysaccharides in a preparation process, higher mechanical strength and stability of a final product, and higher mass transfer performance and pelletizing performance. The fresh water composite microorganism base modifier modifies culturing sewage through the three strong effects of fluctuation, enzymatic degradation and microbial degradation, and has the advantages of comprehensive biodegradability, high security, nontoxicity, harmlessness and freeness from secondary pollution.