Two-photon microscopy (TPM) is an indispensable bioimaging tool. TPM, which uses photons of a near-infrared wavelength as an excitation source for fluorophores, can result in deeper tissue images, which can potentially be measured beyond a depth of 1000 µm with relative stability in biological systems. With continuous development in deep-tissue optical microscopy and improvements in imaging algorithms, TPM is gaining immense support in biomedical applications. Furthermore, it has propelled the continuous development of appropriate two-photon (TP) probes with improved sensitivities for measuring target analytes in biological specimens. In this chapter, the photo-physiological process, basic TP probe design strategies, and representative TP sensors of TPM that researchers have reported are presented to provide significant information regarding specific targets in biological systems.