Early cancer diagnosis is of the most crucial factors determining proper patient management and long-term survival. Thus, there is considerable interest in developing more reliable methods for cancer diagnosis. Fluorescence diagnosis using metabolic alterations of neoplasia has emerged as a powerful new biomedical tool. It is characterized by superior sensitivity and selectivity over other methods, and it offers the possibility to obtain molecular information required to determine the state of the disease. Furthermore, important advancements have been made in generating deeper three-dimensional images in vivo by exploiting tissue transparency in the near-infrared region and fluorescence-mediated tomography, which might render fluorescence diagnosis less invasive. From basic principles in optics and molecular biology to the development of medical instrumentation and the launching of clinical trials, we have comprehensively compiled the basic information required to clearly understand fundamental aspects of fluorescence-based diagnosis.