This paper presents bio-oil preparation by direct liquefaction of Dunaliella tertiolecta (D. tertiolecta) with sub/supercritical ethanol-water as the medium in a batch autoclave with high temperature and high pressure. The results indicated that ethanol and water showed synergistic effects on direct liquefaction of D. tertiolecta. The maximum bio-oil yield was 64.68%, with an optimal D. tertiolecta conversion of 98.24% in sub/supercritical ethanol-water. The detailed chemical compositional analysis of the bio-oil was performed using an EA, FT-IR, and GC-MS. The empirical formulas of the bio-oil obtained using the ethanol-water co-solvent (40%, v/v) and sole water as the reaction medium were CH15.2O0.14N0.06 and CH1.43O0.23N0.09. with calorific values of 34.96 and 29.80 MJ kg(-1), respectively. XPS and SEM results showed that ethanol-water is a very effective reaction medium in the liquefaction. A plausible reaction mechanism of the main chemical component in D. tertiolecta is proposed based on our results and the literatures. Crown Copyright (C) 2012 Published by Elsevier Ltd. All rights reserved.