Binaphthyl-based chiral fluorescent probes (S)-/(R)-1 featuring a 2-methylthiophenimide unit were designed and synthesized.The fluorescent response to enantiomer pairs of amino acids revealed that (S)- and (R)-enantimer of the probe had chiral bias to L- and D-amino acids, resp.The compound (S)-1 showed outstanding enantioselective fluorescence enhancement toward 10 amino acids (Asn, Glu, Val, Leu, Met, Ser, Gln, Arg, Phe and Ala) in both solvents of EtOH/H2O and DMSO/H2O at the condition of pH 7.5.Extremely high enantiomeric fluorescence enhancement ratio (ef, [IL-I0]/[ID-I0]) were obtained for Asn (646.2), Glu (318.9), Val (255.6), Leu (252.4), Thr (223.9) in EtOH/H2O, and the ef values to Val, Ala, Ser, Glu, Arg, Gln were all larger than 20 in DMSO/H2O.Chiral fluorescent imaging of the four amino acids (Ser, Val, Arg and Gln) were obtained in both living cells and zebrafishes via the probe (R)-1, which provides a new concept for the diagnosis and treatment of diseases associated with amino acid racemization.Results of 1H NMR and HRMS revealed the enantioselective fluorescence enhancement mechanism.