The harvesting of microalgae biomass from culture medium is a tedious process that needs high cost and energy. Calcium chloride was utilized as a flocculant for the flocculation process of Chlorella pyrenoidosa in an alkaline medium. This study was aimed to optimize the four flocculation process parameters for microalgae by Box-Behnken design of Response Surface Methodology. The maximum flocculation efficiency of C. pyrenoidosa was obtained 97.58% at 32.5 °C, pH 9, 37.5 min. and 275 mgL −1 flocculant dose, out of which 72 (± 2) % of flocculant amount was contributed in the flocculation process. The rest of the 28% flocculant was recovered from the spent medium, which was again utilized for the flocculation process and was achieved 28% flocculation efficiency at pre-optimized conditions. The cost of the flocculation process was analyzed ≈0.10 USD for 1 kg of microalgae biomass which shows a low-cost option compared to conventional chemicals. Graphical abstract