Abstract: Two-photon fluorescence dyes have shown promising applications in biomedical imaging. However, the substitution site effect on geometric structures and photophysical properties of fluorescence dyes is rarely illustrated in detail. In this work, a series of new lipid droplets detection dyes are designed and studied, molecular optical properties and non-radiative transitions are analyzed. The intramolecular weak interaction and electron-hole analysis reveal its inner mechanisms. All dyes are proven to possess excellent photophysical properties with high fluorescence quantum efficiency and large stokes shift as well as remarkable two-photon absorption cross section. Our work reasonably elucidates the experimental measurements and the effects of substitution site on two-photon absorption and excited states properties of lipid droplets detection NAPBr dyes are highlighted, which could provide a theoretical perspective for designing efficient organic dyes for lipid droplets detection in biology and medicine fields.