Fluorescence lifetime is the average amount time a fluorophore spends in excited state before returning to the ground state. In this paper, methods for quantifying intra- and inter-molecular interactions based on fluorescence lifetime measurement were developed. One is quantification of DNA secondary structures formation by fluorescence lifetime change through G-base quenching, and the other is quantification of inter-molecular interactions by fluorescence lifetime change through fluorescence resonance energy transfer (FRET). The first method cleverly utilizes the properties of G-base to quench adjacent dye molecules, and combines with changes in fluorescence lifetime, to determine the formation of DNA secondary structure and the formation ratio. FRET is an important means for studying biological molecular interactions. Traditional FRET methods are mainly based on change in intensity, but this change is susceptible to changes in fluorescence expression levels, molecular diffusion in samples and crosstalk between fluorophores, which brings complexity in experimental design and poor repeatability of experimental data. FRET measurement based on fluorescence lifetime can overcome the disadvantages mentioned above. By detecting changes in the fluorescence lifetime of the donor, we can quickly and easily determine whether FRET occurs, and through establishing a systematic data analysis method we can get FRET efficiency and information about inter-molecular interactions.