Chlorophyll acts as a beneficial antioxidant for the human body; however, only a few studies have reported the chlorophyll extraction using the non-toxic solvents. In this study, we used a green solvent, 2,3-butanediol (2,3BDO), for extraction of chlorophyll a from Nannochloropsis sp. for the first time. Levo-isomer of 2,3-BDO exhibited remarkable chlorophyll extraction properties compared to its meso-isomer, and other conventional solvents such as ethanol. Hydrogen bonding between the solvent and chlorophyll was a detrimental factor for evaluating the extraction efficiency. The extraction yield reached 91.8% with the maximum specific antioxidant activity at 10,000 rpm for 20 min, using 25 mL solvent/g dry cell with assistance of a high shear mixer (HSM). The scalability was verified upon comparing the lab- and bench-scale tests, which demonstrated similar extraction yields and antioxidant activities, reducing process time and specific energy consumption.