With the development of modern energy, the use of clean and renewable new energy has gradually become the main idea to solve environmental and energy problems. Among the many new energy sources, biomass energy not only reduces greenhouse gas emissions, but also has good renewable properties. Microalgae is an ideal preparation of biomass raw materials which can grow fast and are rich in oil. However, their density are close to water making harvesting difficult. Harvesting difficulty is one of the main constraints on the development of the microalgae industry. In this study, a flotation method based on bead flotation is using a re-frying oil emulsion as flotation beads for harvesting Chlorella vulgaris. The re-frying oil will be fully emulsified with pure water in a certain proportion under the action of emulsifier. Combining with the response surface method, the significant factors affecting the harvesting efficiency were screened as pH, stirring rate, and proportion of protoalgae; the significant factors affecting the enrichment ratio were pH, flotation time, and proportion of protoalgae. The optimal harvesting efficiency and enrichment ratio were obtained as 92.79% and 2.07%, respectively. By taking Chlorella vulgaris as the research object, using XDLVO theory (extended Derjaguin-Laudau-Verwey-Overbeek), the mechanism of action between re-frying oil floating beads and Chlorella vulgaris was discussed. The mechanism of harvesting microalgae by re-frying is that microalgae and buoy beads form aggregates by electric neutralization and bridging due to of aluminum ion. The microalgae and the buoy beads first approach each other under the action of kinetic energy provided by stirring, and then by van der Waals force to adhere. The gravitational action reaches the critical value at 14.73 nm. The harvesting cost is only 4.38 ￥/t under the optimal harvesting conditions. This method reduces the harvesting cost and energy consumption which provides a method for the reuse of re-frying oil.