The invention discloses a preparation method of biological diesel. The method comprises the following steps: adding methanol and raw oil (various preprocessed greases) into a reaction kettle in the mol ratio of (4:1)-(8:1), adding acid, and carrying out pre-esterification under negative pressure; adding water to neutralize and wash, dehydrating, removing impurities, drying, sending into the reaction kettle, heating to 75 DEG C, adding alkali, and carrying out transesterification under negative pressure; separating out the glycerol methanol solution on the lower layer to obtain crude biological diesel; and carrying out tertiary molecular distillation to obtain fatty acid methyl ester containing C16, C18 and C20, thereby obtaining the biological diesel. The invention has the advantages that the method is simple and the overall esterification time is shortened by 1/2; the transesterification rate is higher than 93%; the dehydration, decoloring, degassing and other working procedures of the intermediate products are completed in one step, thereby shortening the process, reducing the energy consumption and lowering the cost; and the obtained product has stable quality, and all the technical indices achieve and even exceed the 0# diesel national standard.