Linear photophysical characterization and two-photon absorption (2PA) properties of a new water-soluble fluorene derivative, 3-(9-(2-(2-methoxyethoxy)ethyl)-2,7-bis{3-[2-(polyethyleneglycol-550-monomethylether-1-yl)]-4-(benzo[d]thiazol-2-yl)styryl}-9H-fluoren-9-yl)propanoic acid (1), were investigated in several organic solvents and water at room temperature. A comprehensive analysis of the steady-state absorption, emission and excitation anisotropy spectra revealed electronic structures of 1, including mutual orientation of the transition dipoles, relatively weak solvatochromic effects, high fluorescence quantum yield (approximately 0.5-1.0), and strong aggregation in water. The 2PA spectra of 1 were obtained in the 600-900 nm spectral range by two-photon induced fluorescence (2PF) and open aperture Z-scan methods using femtosecond laser sources. No discrete 2PA bands were apparent and values of the corresponding 2PA cross sections monotonically increased in the short wavelength range up to 3000 GM in organic solvents and approximately 6000 GM in aqueous solution, reflecting relatively high two-photon absorptivity. The 2PA efficiency of in water increased 2-3 times relative to aprotic solvents and can be explained by cooperative electronic effects of molecular aggregates of 1 produced in aqueous media. The carboxylic acid fluorenyl probe 1 was conjugated with the cyclic peptide RGDfK. Two-photon fluorescence microscopy (2PFM) imaging of U87MG cells (and MCF-7 as control), incubated with fluorene-RGD peptide conjugate 2, demonstrated high alpha(v)beta(3) integrin selectivity, making this probe particularly attractive for integrin imaging.