Excessive of carbon dioxide (CO2) emission and water pollution have been identified as the two primary challenges to humans and environment. Hence, biological carbon sequestration by microalgae is recommended as an environmentally friendly approach to capture and convert this CO2 into value-added products. However, research related to the development of efficient system to concurrently overcome low CO2 solubility in water and reduction of water footprint in microalgae cultivation is still limited in the literature. In this study, the CO2 capture by Chlorella vulgaris in a recycled cultivation medium was exploited using a sequential flow photobioreactor system. The study revealed that nutrient replenished recycled medium did not significantly affect the growth performance and lipid content of C. vulgaris. It was also observed that the CO2 capture efficiency and protein content were gradually increased from the first (SFB-RWN1) to the third (SFB-RWN3) cycle of cultivation due to the increment of carbon and nitrogen content in the microalgae cell. Besides, the lipid profile of C. vulgaris cultivated in the recycled medium comprised of high concentration of saturated (up to 32.41%) and polyunsaturated (up to 43.21%) fatty acid methyl ester (FAME). The present study suggested that growing C. vulgaris in a recycled medium is a feasible solution to fix CO2 from the atmosphere and help to reduce water footprint in the microalgae cultivation system.