Efficient reversible phototransformation of a new diarylethene-fluorene derivative, 1,2-bis(5-(9,9-didecyl-7-nitro-9H-fluoren-2-yl)-2-methylthiophen-3-yl)cyclopent-1-ene (1), was demonstrated in organic media under low-intensity laser excitation. Linear photophysical characterization of 1 was performed at room temperature in solvents of different polarity and viscosity. Significantly, close to unity quantum yield for the cyclization reaction of 1 was shown in nonpolar solutions. The lifetimes of the excited states of the open (OF) and closed (CF) forms of 1 were measured by a femtosecond transient absorption technique, and corresponding values of ∼0.7 and ∼0.9 ps were shown in dichloromethane (DCM), respectively. Degenerate two-photon absorption (2PA) spectra of the OF and CF of 1 were obtained over a broad spectral range by the open aperture Z-scan method under 1 kHz femtosecond excitation. The values of 2PA cross sections of the OF in DCM (∼50-70 GM) were found to increase up to 1 order of magnitude (∼600 GM) after cyclization to the CF. The nature of cyclization and cylcoreversion processes were investigated by quantum chemistry with employment of DFT-based methods implemented in the Gaussian'09 program. The potential of 1 for application in optical data storage was shown using poly(methyl methacrylate)-doped films and two-photon fluorescence microscopy readout.