Biodiesel production from Scenedesmus sp. was optimized through in situ transesterification via acid catalysis associated with high pressure homogenization pretreatment. Response Surface Methodogy (RSM) was applied to investigate the influence of catalyst amount and biomass to solvent ratio as two major factors for in situ transesterification by central composite method. Optimized reaction conditions were determined as H 2 SO 4 amount of 5.46 % and biomass to methanol ratio of 1:22.07 at 70 ºC for 10 hrs reaction time. The Fatty Acid Methyl Ester (FAME) from this process was confirmed to be almost identical to a commercial standard biodiesel by TLC analysis. Biodiesel yield from the quadratic response surface model was also calculated as 69.36 % (base on lipid weight) of the maximum yield under these conditions, and this theoretical yield showed good agreement with 69.11±1.16 % of experimental result. From GC analysis of biodiesel, ten species of C 16 -C 22 with saturated and unsaturated fatty acid were identified, and predominate fatty acids were C 16 and C 18 methyl esters. These results confirm that an efficient production of biodiesel from mciroalge, Scenedesmus sp. could be possible through an optimized in situ acidic transesterification process.