The steady-state photophysical, NMR, and two-photon absorption (2PA) properties of a new fluorene derivative (1) containing the 2-(2'-hydroxyphenyl)benzothiazole (HBT) terminal construct is investigated for use as a fluorescence probe in bioimaging. A comprehensive analysis of the linear spectral properties reveals inter- and intramolecular hydrogen bonding and excited state intramolecular proton transfer (ESIPT) processes in the HBT substituent. A specific electronic model with a double minimum potential energy surface is consistent with the observed spectral properties. The 2PA spectra are obtained using a standard two-photon induced fluorescence method with a femtosecond kHz laser system, affording a maximum 2PA cross section of approximately 600 GM, a sufficiently high value for two-photon fluorescence imaging. No dependence of two-photon absorption efficiency on solvent properties and hydrogen bonding in the HBT substituent is observed. The potential use of this fluorenyl probe in bioimaging is demonstrated via one- and two-photon fluorescence imaging of COS-7 cells.