Nanobiotechnology is one of the fastest growing and broadest-ranged interdisciplinary subfields of the nanosciences. Countless hybrid bio-inorganic composites are currently being pursued for various uses, including sensors for medical and diagnostic applications, light- and energy-harvesting devices, along with multifunctional architectures for electronics and advanced drug-delivery. Although many disparate biological and nanoscale materials will ultimately be utilized as the functional building blocks to create these devices, a common element found among a large proportion is that they exert or interact with light. Clearly continuing development will rely heavily on incorporating many different types of fluorophores into these composite materials. This review covers the growing utility of different classes of fluorophores in nanobiotechnology, from both a photophysical and a chemical perspective. For each major structural or functional class of fluorescent probe, several representative applications are provided, and the necessary technological background for acquiring the desired nano-bioanalytical information are presented.