This chapter first reviews the fundamental aspects of the two-photon absorbing materials developed in our group. The design strategies and syntheses of a variety of fluorene-based conjugated molecules as two-photon absorbing materials, along with their photochemical and photophysical properties are described. The methodology of synthesis of these 2PA chromophores designed with various donor or acceptor groups, conjugation lengths and symmetries and the effects of the structure on the 2PA properties are demonstrated. This is followed by presentation of detailed studies of their linear absorption, steady-state and time-resolved fluorescence, fluorescence life time and anisotropy, excited-state absorption, and two-photon absorption measurements. The photostabilities of these chromophores are also investigated due to importance of this parameter in several emerging applications. The last part of the chapter provides a description of the application of these materials in fluorescence imaging, 3D data storage, photodynamic therapy, and 3D microfabrication.