Fluorescence spectroscopy and imaging have been widely used for in vivo cancer diagnosis and therapy monitoring in preclinical models, as well as clinical translation. Great attempts have been made to develop novel fluorescence techniques and improve on existing ones, which can now be used in conjunction with newly developed fluorescent probes for specific cancer imaging. In this review, a broad overview of fluorescence techniques is provided, including photodynamic diagnosis, laser confocal endomicroscopy and fluorescence lifetime imaging, coupled with endogenous and exogenous fluorophores. In particular, endogenous fluorophores, such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), are highlighted as they are linked to cellular metabolism in precancer growth. The use of near-infrared dyes, such as indocynanine green (ICG), for imaging deep-tissue regions is also reviewed. In addition, diagnostic algorithms used for tissue classification and cancer detection will be discussed. Lastly, emerging technologies in fluorescence diagnosis will also be included.