Abstract: MgFe-hydrotalcites (MgFe-LDH) were prepared by using improved urea method and used as the catalysts in the synthesis of biodiesel oil from chlorella protothecoides oil. The hydrotalcites were characterized by XRD, FT-IR, SEM as well as Fourier self-deconvolution and band curve-fitting analyses; the effects of pH value, Mg/Fe mol ratio, temperature and time to prepare the hydrotalcites on their properties and catalytic performances were investigated. The results showed that the MgFe-LDH with Mg/Fe mol ratio of 3 and 4 (Mg3Fe and Mg4Fe), prepared at 110 ℃ and pH value of 9.5 for 10 h exhibits high symmetrical crystalline structure. The brucite-like layer of Mg3Fe and Mg4Fe hydrotalcites are stable under calcination for a completely decomposition. compared with the Mg2Fe hydrotalcite with Mg/Fe mol ratio of 2, Mg3Fe and Mg4Fe upon calcination exhibit higher crystallinity and more active sites and higher catalytic activity. For the synthesis of biodiesel oil from chlorella protothecoides oil over the Mg3Fe catalyst, with methanol/oil molar ratio of 6 in the feed, the biodiesel oil yield reaches 87% after reaction 1.5 h; the catalyst can be reused for three times without serious deactivation.