Abstract: We used the method of GC-MS to analysis the composition and content of the fatty acid in oil-rich Isochrysis sp. CCMM5001 to prepare bio-diesel, and determined the characteristics of bio-diesel such as density, viscosity, flash point 、acid value and degree of copper corrosion. The result showed that fatty acids in the oil-rich Isochrysis sp. CCMM5001 are mainly C16, C18 and C22, total fatty acid content is 82.93 \% in crude lipid; the main content of the prepared bio-diesel is C16 and C18 fatty acid methyl esters, which is 93 \% of the total product. The density, viscosity, flash point, acid value and degree of copper corrosion of the prepared bio-diesel basically conform to the standard of market circulation. The experiment showed that using oil-rich Isochrysis sp. CCMM5001 which has higher fatty acids content as a raw material for preparation of bio-diesel is significant, and needs further study. The prepared bio-diesel can be an ideal substitute for prevailing diesel in the market.