The invention discloses a preparation method of anti-algae room temperature vulcanized silicone rubber (RTV). The method comprises the following steps: S1, weighing a certain mass of anti-microbial agent powder and silicon dioxide powder according to a required mass ratio and physically mixing to obtain a powdery mixture; S2, placing the powdery mixture into a closed container and uniformly heating; starting to time when the heating temperature reaches the melting point of the anti-microbial agent, and stopping heating after at least 30 minutes; S3, cooling the obtained mixture to room temperature so as to obtain a curd-containing mixture; then, dispersing the curd-containing mixture, weighing the dispersed mixture with preset mass according to a given mass fraction of the anti-microbial agent, adding into an RTV coating, and then mixing; S4, drying and vulcanizing the RTV coating added with the mixture at room temperature so as to form the solid silicon rubber.