Currently, there is no effective way to assess the therapeutic response of temozolomide (TMZ) for the glioma. In this study, the human U87MG-fLuc glioma animal models were set up and the antitumor efficacy of TMZ was evaluated using bioluminescence imaging (BLI) and MRI. Then, bioluminescence tomography (BLT) was reconstructed using an adaptive sparsity matching pursuit (ASMP) algorithm. Second, the expression level of the MMP-750 probe was examined with or without TMZ treatment using FMI. Third, the expression of MMP2 and MMP3 was specifically examined after treatment. The results showed that TMZ effectively inhibited glioma growth. The targeted imaging of MMP-750 was decreased during the treatment of glioma with TMZ. Moreover, the MMP2 and MMP3 expression was found to correlate with the inhibition effect of TMZ. Our study indicated that the therapeutic effects of TMZ can be effectively evaluated at an early stage using molecular imaging, and MMP targeting the fluorescence probe could be utilized for the prediction and assessment of the therapeutic effects of TMZ.