The most common intracranial malignant tumors (e.g., gliomas and metastatic tumors often present infiltrative growth, unclear boundaries, low rate of surgical resection, and high surgical risk. How to determine the tumor boundaries during the operation is crucial for surgical resection of the tumor and the protection of brain function. This chapter introduces the recent studies of applications of fluorescence probes in illuminating tumor margins and protect functional area where traditional white light imaging fails to do so.