Nanomaterials with unique optical properties and biocompatibility have been widely employed for designing and fabricating highly selective and sensitive nanosensors for the detection of various chemical and biological species. The development of nanomaterial-based chemo- and biosensors is studied usually under direct spectroscopic and reagent-mediated sensor platforms using both unmodified and surface-functionalized nanomaterials. This chapter mainly focuses on selective sensing of chemical and biological molecules using various types of nanomaterials. The main readouts are absorption (colorimetric, UV-visible), fluorescence, Raman/SERS spectroscopic, and electrochemical sensing techniques. The detailed discussion on the design of nanomaterial-based sensing systems, sensing principle, sensing method, and their signaling mechanisms has been provided. The sensing systems can also be ideally utilized for real-time applications.