As a high-sensitivity imaging modality, bioluminescence tomography can reconstruct the three-dimensional (3-D) location of an internal luminescent source based on the 3-D surface light distribution. However, we can only get the multi-orientation two-dimensional (2-D) bioluminescence distribution in the experiments. Therefore, developing an accurate universal registration method is essential for following bioluminescent source reconstruction. We can then map the multi-orientation 2-D bioluminescence distribution to the 3-D surface derived from anatomical information with it. We propose a 2-D -to-3-D registration method based on iterated optimal projection and applied it in a registration and reconstruction study of three transgenic mice. Compared with traditional registration methods based on the fixed points, our method was independent of the markers and the registration accuracy of the three experiments was improved by 0.3, 0.5, and 0.4 pixels, respectively. In addition, based on the above two registration results using the two registration methods, we reconstructed the 3-D location of the inner bioluminescent source in the three transgenic mice. The reconstruction results showed that the average error distance between the center of the reconstructed element and the center of the real element were reduced by 0.32, 0.48, and 0.39 mm, respectively.