Purpose: Malignant gliomas are major causes of cancer-related mortality and morbidity. Traditional surgery usually leads to incomplete resection of gliomas resulting in the high incidence of tumor recurrence. Advanced medical imaging technology, such as fluorescence imaging-guided surgery, combined with tumor-specific imaging probes allows the identification of tumor margins and improved surgery. However, there are two pressing issues that need to be addressed: first, few fluorescence imaging probes can specifically target gliomas; second, fluorescence molecular imaging (FMI) cannot get the in-depth information of deep-seated gliomas; both of which affect the complete removal of the gliomas. Procedures: In this study, the biodistribution of smart matrix metalloproteinase (MMP) targeting near-infrared (NIR) fluorescent probe MMPSense 750 FAST (MMP-750) was examined in both U87MG-GFP-fLuc glioma xenograft and orthotopic mouse models using FMI. Then, CT and FMI images of orthotopic gliomas were acquired for the reconstruction of fluorescence molecular tomography (FMT) using a randomly enhanced adaptive subspace pursuit (REASP) algorithm. Furthermore, the resection of orthotopic glioma was performed using the fluorescence surgical navigation system after the injection of the MMP-750 probe. After surgery, bioluminescence imaging (BLI) and hematoxylin and eosin staining were carried out to confirm the precision resection of the tumor. Results: FMI results showed that the MMP-750 probe can specifically target U87MG glioma in vivo. FMT presented the spatial information of the orthotopic glioma using the REASP reconstruction algorithm. Furthermore, MMP-750 could effectively delineate the tumor margin during glioma surgery leading to a complete resection of the tumors.