The optical constant of biodiesel plays an important role in quantitatively and qualitatively analyzing its individual constituent, judging the mixed level with diesel fuel, and studying the intrinsic mechanism of radiation transfer. Transmittance model of the three-layer-plate structure (optical glass -biodiesel -optical glass) is derived based on the ray trace method. Experimental measurement by double optical pathlength transmission method is conducted to study the temperature dependence of optical constant of biodiesel in the spectral range from 300 nm to 2500 nm. The results show that the refractive index of biodiesel decreases linearly with temperature increasing from 20 ℃ to 60 ℃, but the absorption index is less affected. Moreover, five main absorption peaks of biodiesel are observed at 1200, 1390, 1750, 2150 and 2300 nm, which are due to overtones and combinations of C—H stretching vibration of various chemical groups. Compared with ordinary vegetable oil, absorption peaks of the biodiesel exhibit the same locations but different amplitudes.