A reconstruction method is presented for spectrally resolved three-dimensional bioluminescence tomography (BLT) in heterogeneous media using a level-set strategy. In order to reconstruct internal bioluminescent sources, a level-set strategy is utilized to quantitatively localize the distribution of bioluminescent sources. The results in numerical phantom experiments clearly show that the proposed method can tolerate different initial values and noise levels and, furthermore, can work credibly even when the number of phases (levels) is not known a priori. In addition, a mouse atlas reconstruction is employed to demonstrate the effectiveness of the proposed method in turbid mouse geometry. Finally, the physical experiment further evaluates the method's potential in practical applications.