Seven new donor–spacer–acceptor metal-free organic dyes containing a fluorene and/or thiophene unit as a conjugated spacer are prepared and their physicochemical properties as well as photovoltaic performance are studied. The electron transfer from donor to acceptor upon excitation of these dyes is sensitive to the conjugated spacers. If a stronger donor moiety such as (ethylenedioxy)thiophene is used as a spacer, such as in F-E1 dye, the dye molecule has a better charge separation and therefore high efficiency. Furthermore, for the same donor and acceptor, fluorene is a better conjugated spacer than the bithiophene moiety because of its nonplanar conformation, which avoids the electron–hole recombination of dye upon photoexcitation. Short molecules with planar conformation aggregate easily, and the corresponding device reveals low open-circuit voltage Voc. On the other hand, dye molecules with long alkyl side chains (large size) adsorb less on the TiO2 electrode, which results in a smaller short-circuit current density Jsc. The difference in the efficiency of these dyes is small except for F-1 (owing to its short conjugation length), and electron density difference map analyses reveal that photoexcitation transfer of electron density from the donating moieties toward the acceptor/anchoring units occurs for all molecules. The small difference in the efficiency is attributed partly to the degree of effective charge transfer upon excitation, not totally to the absorption profile of the dye molecules. Furthermore, time-dependent DFT simulated absorption spectra are in excellent agreement with experimental spectra when the dyes are in their protonated states in THF solution. The transition probability calculated with the electron density difference analyses combined with the absorption data and photovoltaic performance provide useful information for designing organic sensitizers for dye-sensitized solar cells.