This study is to report a ZnSe quantum dot with a large two-photon absorption cross section and good biocompatibility, which can be used in bioimaging. Fluorescence emission at 410 nm is observed in the quantum dot under 760-nm laser excitation. These biocompatible quantum dots exhibit a two-photon cross-section of 9.1 × 105 GM (1 GM = 10−50 cm4⋅s/photon). Two-photon excited laser scanning microscopic images show that cells co-cultured with ZnSe quantum dots are found in the blue channel at a fluorescence intensity that is 14.5 times that of control cells not co-cultured with quantum dots. After incubating zebrafish larvae with ZnSe quantum dots for 24 h, the fluorescence intensity of the yolk sac stimulated by ultraviolet light is 2.9 times that of the control group. The proposed material shows a great potential application in biological imaging.