Fluorescent probes are essential tools for studying biological systems. The last decade has witnessed particular interest in the development of two-photon excitable probes, owing to their advantageous features in tissue imaging compared to the corresponding one-photon probes. This review summarizes various types of two-photon probes that have been applied to bioimaging, categorized by the principles in the probe design and the target analytes, which would provide a basis for the future development of novel two-photon probes for tackling important biological issues.