The degenerate two-photon absorption (2PA) spectra of several fluorene-based photosensitizers (PS) in solution were obtained over a broad spectral range (460−880 nm) by open aperture Z-scan and two-photon fluorescence methods under either picosecond or femtosecond excitation, respectively. A maximum 2PA cross section of ca. 300 GM was observed for the photosensitizers containing a benzothiazole substituent in the fluorenyl 7-position. The electronic structures and 2PA properties of these PS were analyzed using a time-dependent density functional theory method, resulting in reasonably good agreement between experimental and theoretical data.