In order to efficiently separate large-molecules and high-viscosity lipids from microalgal biomass without harmful solvents, a solvent-free lipid extraction method with subcritical water is proposed to break hydrogen bonds and hydrolyze microalgal lipids in a continuous flow reactor for acid-catalyzed biodiesel production. Crude oils were efficiently extracted from microalgal biomass with subcritical water and were characterized by elemental analysis, Fourier transform infrared spectroscopy and thermogravimetric analysis. The solvent-free extraction efficiency from microalgal biomass treated with subcritical water at 260 degrees C and the percentage of crude oil capable of esterification, dramatically reached 72.3% and 44%, respectively. The acid-catalyzed esterification efficiency of crude oil extracted from microalgal biomass with subcritical water at 260 degrees C reached 98.4%, as compared to 48.4% for microalgal lipids extracted through a traditional process.