The invention discloses hexa-substituted coumarin derivative two-photon fluorescent dye of which the structural general formula is shown in the specification, wherein R is CH3O- or (CH3)2N-. The fluorescent dye prepared by the invention is simple to synthesize, obtains a monocrystalline structure, has an effective two-photon absorption cross section, can generate fluorescence through two-photon excitation, has low cytotoxicity, and can enter a cell and be used for biological imaging of the cell.