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Surgeons need surgical navigation and intraoperative imaging for accurate surgery. However, current clinical surgical navigation systems have many drawbacks, including the need of fiducial markers, the lack of real-time registration update, and lack of functional imaging. We have developed a novel Real-time Fluorescence Imaging Topography Scanning (RFITS) system that delivers simultaneous real-time multimodal optical imaging, CT-to-Optical image registration, and dynamic fiducial-free surgical navigation. The RFITS prototype was characterized to evaluate system performance and used in biological models for fluorescence-guided surgery. We also performed multimodal imaging of a casting life-sized human cranial model and investigated image registration between intraoperative optical image data and preoperative tomographic image data. The RFITS prototype successfully integrated real-time intraoperative optical imaging and dynamic CT-based surgical navigation, offering complementary functional and structural information for surgical guidance. Our results demonstrated that the RFITS system can provide accurate multimodal surgical guidance in preclinical studies. To the best of our knowledge, this is the first report of a system capable of concurrent intraoperative fluorescence imaging and dynamic, real-time, CT-based navigation, whereas multiple modalities are accurately registered.