A water-dispersible PEGylated conjugated oligomer (PEGTVFVBN) is synthesized for two-photon fluorescence imaging applications. PEGTVFVBN could aggregate into nanoparticles of ~ 110 nm size in H 2 O , which show a fluorescence quantum yield of 0.12, 20 times higher than that for its cationic counterpart (TVFVBC). Two-photon fluorescence measurement reveals a maximum two-photon absorption (TPA) cross-section of 263 GM at 780 nm based on molecules for PEGTVFVBN nanoparticles in H 2 O , while the TPA spectra of TVFVBC are barely detectable under the same experimental conditions. The N , N' -diacetylchitobiotic acid conjugated derivative (ChPEGTVFVBN) could also aggregate into nanoparticles of ~ 90 nm sizes, showing a slightly smaller fluorescence quantum yield of 0.08 and a maximum TPA cross-section of 213 GM at 780 nm. ChPEGTVFVBN could be specifically internalized by vimentin over expressing Hela cells through a receptor mediated endocytosis, giving two-photon fluorescence images in a bright and high contrast manner. In addition, both PEGTVFVBN and ChPEGTVFVBN show low cytotoxicity, which is essential for cell imaging applications. This study thus highlights the molecular engineering strategy of PEGylation to construct robust TPA materials for two-photon fluorescence imaging applications.