Abstract: Provided is a method for preparing a novel benzocoumarin-based two-photon absorbing fluorescent dye, such as a pyridinium-benzocoumarin (Py+BC) derivative compound and its precursor compound (pyridyl-benzocoumarin, PyBC), an aryl-benzocoumarin (ArBC) derivative compound, a benzothiazolyl-benzocoumarin (BtBC) derivative compound or a keto-benzocoumarin (KetoBC) derivative compound. The Py+BC derivative compound of the present invention is a two-photon absorbing near-infrared fluorescent dye, which may minimize interference of autofluorescence compared to conventional two-photon absorbing fluorescent dyes such as acedan and naphthalimide, thereby obtaining a high-definition image, and therefore is expected to be effectively used in imaging studies. In addition, when the two-photon absorbing near-infrared fluorescent dye of the present invention is used, it is expected to be suitable for high resolution imaging of deep tissue.