Single benzene-based fluorophore (SBBF) is a representative small-size fluorescent dye which has an electron push-pull structure within a benzene backbone. In this work, we disclosed a new amino-SBBF (ASBBF) library that has various alkyl-amines and aromatic-amines on the electron-donating site. The synthesis, photophysical properties, quantum chemical calculation, two-photon responsiveness, and bioimaging applicability of ASBBF in cells were successfully identified. We believe that our present work provides a guide to the development of new fluorescent materials and molecular probes in various research fields.