Agal-fermentation-based microbio-diesel production was realized through high-cell-density fermentation of Chlorella protothecoides and efficient transesterification process. Cell density achieved was 16.8 g 1(-1) in 184 h and 51.2 g 1-(1) in 167 h in a 5-1 bioreactor by performing preliminary and improved fed-batch culture strategy, respectively. The lipid content was 57.8, 55.2, and 50.3% of cell dry weight from batch, primary, and improved fed-batch culture in 5-1 bioreactor. Transesterification was catalyzed by immobilized lipase, and the conversion rate reached up to 98%. The properties of biodiesel from Chlorella were comparable to conventional diesel fuel and comply with US standard for Biodiesel. In a word, the approach including high-density fermentation of Chlorella and enzymatic transesterification process were set up and proved to be a promising alternative for biodiesel production.