Lutein is a natural carotenoid with health promoting properties whose commercial source is the yellow Marigold flower, but its production is limited by climate, planting area and high labor costs. The microalga Chlorella vulgaris is a promising lutein source; however, no commercial lutein production uses microalgae. In the present study, the lower phase (LP4) of the biphasic solvent system composed of n-heptane–ethanol–water (5:4:1.5, v/v/v) was used both as a solvent for microalgae biomass extraction and as a mobile phase for lutein isolation using high performance counter-current chromatography (HPCCC). The ultrasound-assisted extraction of biomass with LP4 for 30 min led to an extract with a lutein content (3.20 mg/g dried biomass) that was higher than those obtained by the reference solvents tetrahydrofuran and dichloromethane. A multi-injection HPCCC method was developed for lutein isolation, where LP4 was pumped as the mobile phase at a flow rate of 8 mL/min, whereas its upper phase was the stationary phase. In total, 2 g of C. vulgaris extract were processed affording 60 mg of lutein (92% purity), which was further cleaned up by gel permeation chromatography yielding 50 mg of lutein (97% purity). The chemical identity of the purified compound was confirmed by mass spectrometry and absorption spectroscopy in comparison with an authentic standard. The developed HPCCC method may serve as a reference for promoting the development of a new production model of microalgae-based lutein.