In this study, a recent simple separation technique, three-phase partitioning (TPP), was used for concentrating microalgae Chlorella spp. for the first time. More than 91.7% of the biomass precipitated in the interlayer of the system in 10 min. Temperature, initial concentration and ratio of ethanol to dipotassium hydrogen phosphate (DKP) were observed to negatively correlate with concentration factor while pH showed no significant influences. Using this method, biomass could be concentrated with much lower energy consumption and concentrated biomass could be conveniently collected. Besides, together with concentrating, TPP concentrated microalgae cells showed 26.3% increase in lipid extraction yield. Additionally, similarities in fatty acid profile indicated the avoidance of influence on lipid quality from chemicals. This study demonstrated the feasibility of TPP for microalgae biodiesel production. (C) 2013 Elsevier Ltd. All rights reserved.