The chemical composition of biodiesel was analyzed by gas chromatography-mass spectrometry. The cold filter plugging point of biodiesel was measured according to GB/T 20828-2007. The cold flow properties of biodiesel were investigated on the basis of the crystallization theory, including chemical composition and molecular structure. Biodiesel might be considered a pseudobinary mixture, namely, high-melting-point saturated fatty acid methyl esters and low–melting-point unsaturated ones. Bilayer crystal structure of biodiesel was founded. Bilayer structure with headgroups aligned next to each other inside the crystal and away from nonpolar bulk liquid was large platelet lamellae. The results showed that biodiesel was mainly composed of saturated fatty acid methyl esters (C14:0～C24:0 ) and unsaturated ones(C16:1～C22:1、C18:2 and C18:3 ). The cold flow properties for biodiesel were determined mainly by the amount and molecular structure of saturated fatty acid methyl esters. The long-straight-chain saturated fatty acid methyl esters tend to have relatively poor cold flow properties. The cold flow properties worsen with increasing the amount and carbon chain length of straight-chain saturated fatty acid methyl esters. The ways of adjusting biodiesel composition and treating with depression of cold filter plugging point were given; they could improve cold flow properties.