Bioluminescence tomography (BLT) is a powerful non-invasive molecular imaging tool for in vivo studies of glioma in mice. However, because of the light scattering and resulted ill-posed problems, it is challenging to develop a sufficient reconstruction method, which can accurately locate the tumor and define the tumor morphology in three-dimension. In this paper, we proposed a novel Gaussian weighted Laplace prior (GWLP) regularization method. It considered the variance of the bioluminescence energy between any two voxels inside an organ had a non-linear inverse relationship with their Gaussian distance to solve the over-smoothed tumor morphology in BLT reconstruction. We compared the GWLP with conventional Tikhonov and Laplace regularization methods through various numerical simulations and in vivo orthotopic glioma mouse model experiments. The in vivo magnetic resonance imaging and ex vivo green fluorescent protein images and hematoxylin-eosin stained images of whole head cryoslicing specimens were utilized as gold standards. The results demonstrated that GWLP achieved the highest accuracy in tumor localization and tumor morphology preservation. To the best of our knowledge, this is the first study that achieved such accurate BLT morphological reconstruction of orthotopic glioma without using any segmented tumor structure from any other structural imaging modalities as the prior for reconstruction guidance. This enabled BLT more suitable and practical for in vivo imaging of orthotopic glioma mouse models.