Photodynamic therapy (PDT) has been used clinically for treating various diseases including malignant tumors. The main advantages of PDT over traditional cancer treatments are attributed to the localized effects of the photochemical reactions by selective illumination, which then generate reactive oxygen species and singlet oxygen molecules that lead to cell death. To date, over- or under-treatment still remains one of the major challenges in PDT due to the lack of robust real-time dose monitoring techniques. Time-resolved fluorescence (TRF) provides fluorescence lifetime profiles of the targeted fluorophores. It has been demonstrated that TRF offers supplementary information in drug-molecular interactions and cell responses compared to steady-state intensity acquisition. Moreover, fluorescence lifetime itself is independent of the light path; thus it overcomes the artifacts given by diffused light propagation and detection geometries. TRF in PDT is an emerging approach, and relevant studies to date are scattered. Therefore, this review mainly focuses on summarizing up-to-date TRF studies in PDT, and the effects of PDT dosimetric factors on the measured TRF parameters. From there, potential gaps for clinical translation are also discussed.