Two-photon microscopy (TPM), which uses two photons of lower energy as the excitation source, is a vital tool in biology and clinical science, due to its capacity to image deep inside intact tissues for a long period of time. To make TPM a more versatile tool in biomedical research, we have developed a variety of two-photon probes for specific applications. In this mini review, we will briefly discuss two-photon probes for lipid rafts, lysosomes, mitochondria, and pH, and their biomedical applications.