Design and synthesis of energy transfer dyads-based ratiometric TP fluorescent probe for the ratiometric detection and imaging of various analytes in biological systems is a vital task. A general introduction to molecular engineering of the fluorescence resonance energy transfer (FRET), and through-bond energy transfer (TBET)-based ratiometric TP fluorescent probe for bioimaging applications during the past decade were reviewed. It was suggested that more attention would be committed to development of new FRET/TBET-based ratiometric TP dyads and applications of two-photon fluorescence microscope (TPFM) in areas of bioanalysis and disease diagnosis.