We present a three-dimensional (3D) bioluminescence image reconstruction method with MRI and CT co-registration for small animal molecular imaging. The multi-spectral light intensity distribution of an optical luciferase-luciferin reporter system is measured at the tissue surface of a small animal for the purpose of 3D image reconstruction. The reporter probe distribution inside tissue is calculated with a linear matrix inversion method and a light propagation model based on the simplified spherical harmonics equations. The animal's surface geometry and anatomy is determined from co-registered CT and MR images in order to locate the reconstructed source distribution relative to the animal's anatomy. We present in vivo bioluminescence reconstruction results that demonstrate the performance of our co-registration method.