We prepared amino acid functionalized water-soluble perylene diimides: N,N'-bi(L-glutamic acid)-perylene-3,4;9,10-dicarboxylic diimide (1), N,N'-bi(L-phenylalanine acid)-perylene-3,4;9,10-dicarboxylic diimide (2), N,N'-bi(Lglutamic amine)-perylene-3,4;9,10-dicarboxylic diimide (3) and N,N'-bi(L-phenylalanine amine)-perylene-3,4;9,10-dicarboxylic diimide (4). The structures of 3 and 4 were confirmed by 1H NMR, FT-IR and MS. The maximal absorption bands of compound 1 and 2 in concentrated sulfuric acid were red-shifted for about 48 and 74 nm, respectively, compared with that of Perylene-3,4,9,10-tetracarboxylic acid dianhydride (PTCDA). Nearly no fluorescence was observed for compounds 1 and 2 in water, while compounds 3 and 4 were significantly water-soluble and had very high fluorescent quantum. The mechanism of the optical properties change was discussed, and the π-π stacking caused by H+ led to the changes of fluorescence spectrum and absorption spectrum. The calculated molecular orbital energies and the frontier molecular orbital maps of compounds 1-2 based on density function theory (DFT) calculations were reported. Owing to the high water-soluble, the perylene derivatives 3 and 4 were successfully applied as high-performance fluorochromes for living hela cells imaging.