To improve microalgae harvesting efficiency and to reduce the addition of chemicals in the buoy-bead flotation process, a novel buoy-bead flotation approach has been developed for harvesting Chlorella vulgaris, using surface-layered polymeric microspheres (SLPMs). Next, the detachment of microalgae cell-SLPM aggregates and the reusability of SLPMs were investigated. The experimental results showed that a maximum harvesting efficiency of 98.43% was achieved at a SLPM dosage of 0.7 g/L and a pH of 9, and harvesting efficiency quickly decreased with increasing ionic strength. A detachment efficiency of 78.46% and a concentration factor of 19.56 were achieved at an ionic strength of 700 mM and a mixing speed of 3000 rpm without changing the pH. Reused SLPMs can still reach an efficiency of 72.13% after five cycles. The presented results show that this method can potentially be applied for large-scale microalgae harvesting.