In this work, we report the synthesis of a family of donor-acceptor (D-A) π-conjugated aggregation-induced red emission materials (TPABT, DTPABT, TPEBT and DTPEBT) with the same core 2,2-(2,2-diphenylethene-1,1-diyl)dithiophene (DPDT) and different amounts and different strengths of electron-donating terminal moieties. Interestingly, TPABT and TPEBT, which have asymmetric structures, give obviously higher solid fluorescence quantum efficiencies in comparison with those of the corresponding symmetric structures, DTPABT and DTPEBT, respectively. In particular, the thin film of TPEBT exhibited the highest fluorescence quantum efficiency of ca. 38% with the highest αAIE. Moreover, TPEBT and DTPEBT with TPE groups showed two-photon absorption cross-sections of (δ) 1.75 × 103 GM and 1.94 × 103 GM at 780 nm, respectively, which are obviously higher than the other two red fluorescent materials with triphenylamine groups. Then, the one-photon and two-photon fluorescence imaging of MCF-7 breast cancer cells and Hela cells, and cytotoxicity experiments, were carried out with these red fluorescent materials. Intense intracellular red fluorescence was observed for all the molecules using one-photon excitation and for TPABT using two-photon excitation in the cell cytoplasm. Finally, TPEBT is biocompatible and functions well in mouse brain blood vascular visualization. It is indicated that these materials can be used as a specific stain fluorescent probe for live cell imaging.