Bioluminescence tomography （BLT） is a promising in vivo molecular imaging tool that allows non-invasive monitoring of physiological and pathological processes at the cellular and molecular levels. The reconstruction accuracy of BLT is affected by the optical transmission model error and the ill-posedness of the inverse problem. The higher-order optical transmission model was able to improve the precision， while the multi-spectral method was able to alleviate the ill-posedness of the inverse problem. In this study， the spectral differential strategy， combining the spectral differential theory and multi-spectral method， was applied on the optical transmission model based on the diffusion approximation equation （DE） and third simplified spherical harmonic approximation equation （SP₃）. First， errors in these two radiative transfer equations （RTE） approximations were analyzed， and the attenuation effect on the error was compared when the spectral differential strategy was applied on two types of optical transmission models. The forward simulation experiment results showed that the spectral differential strategy can effectively reduce the model error of the DE and SP₃ models. The spectral differential strategy resulted in the transmission accuracy of the DE model resembling that of the SP₃ model， and decreased the high requirements on computing time and storage space of high-order approximation. On this basis， the spectral differential strategy was applied on the DE and SP₃ optical transmission models for light source reconstruction. The experimental results showed that the spectral differential strategy not only improves the accuracy of the two light transmission models， but also alleviates the ill-condition of the inverse problem in BLT， yields a location error of reconstructed source within 1 mm， and improves the accuracy of the light source reconstruction in target location， shape restoration， and image contrast. The average time required by the SP₃ model was approximately 1 525 s. In contrast， the DE model combined with spectral differential strategy had an average time consumption of only approximately 34 s， resulting in balanced reconstruction accuracy and speed.