Two dicyanostilbene-derived two-photon fluorescence (TPF) dyes with a D-π-A architecture were synthesized as the model compounds to systematically study the effect of the dicyano and the terminal substituent on the two-photon absorption (TPA). These two compounds (DMO and DMA) exhibit very large two-photon absorption cross sections (δ). DMA with the terminal dimethylamino group has longer emission wavelength (λem(DMA) = 554 nm > λem(DMO) = 459 nm), larger δ (δ(DMA) = 3650 GM > δ(DMO) = 830 GM) and higher fluorescence quantum yield (η(DMA) = 0.456 > η(DMO) = 0.294) than with the terminal methoxy group. The ideal terminal substituent should not be the alkoxy group but the substitutedamino group. This class of dicyanostilbene dyes possess small molecule size, large δ (830–3650 GM), long-wavelength emission (459−554 nm) and large Stokes shift (131−150 nm), and are ideal chromophores for TPF labels and probes.