The flocculation efficiency and biomass recovery are key characteristics to consider and measure when researching solid–liquid separation during the harvesting process of Chlorella sp. microalgae. Response surface methodology (RSM) with a central composite design (CCD) is used in conjunction with chitosan as the bio-flocculant to improve the flocculation efficiency and biomass recovery parameters. The pH, bio-flocculant dosage, and cationic inducer dose are all factors that have been determined. A multiple response optimization approach was used to effectively estimate the optimal parameters, which were at pH 8, chitosan concentration of 100 mg/500 mL, cationic inducer concentration of 50 mg/L, and stirring speed of 240 rpm. The overall appropriateness function approach was used to effectively estimate the optimal parameters. Flocculation efficiency is 96.12%, and biomass recovery is 312.5 mg for the most advantageous flocculation, as well as the flocculation rate was 2.007 min−1. The RSM-CCD approach is beneficial for improving this flocculation process, which will result in a more effective and efficient water reuse process and management of the microalgae harvesting process.