A novel probe (DNSBN) towards biothiols on the basis of 4-hydroxynaphthalimide as fluorophores and 2, 4-dinitrobenzenesulfonyloxy group as specific recognition site was designed and synthesized. The result of absorption and fluorescence spectral analyses indicated that the probe had high sensitivity and selectivity towards cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), and the detection was not affected by other 17 kinds of natural amino acids. Meanwhile, it was confirmed that DNSBN was a ratiometric probe through the fluorescence titration experiment, and the fluorescent intensity at 555 nm had a high linear relationship with biothiols concentration in the range of 0-20 mu mol/L. The detection limits (3 sigma) of Cys, Hcy and GSH were 25. 9, 92. 0 and 77. 9 nmol/L, respectively. The absorption, emission and mass spectra indicated that biothiols could be engaged in nucleophilic substitution reaction with 2, 4- dinitrobenzenesulfonate, which induced the sulfonic esters decomposed. With the departure of receptor unit, the d-PeT progress (donor-excited photoinduced electron transfer) was blocked with an obvious colorimetric and fluorescence change. Finally, HeLa cell imaging experiments verified that DNSBN had good biocompatibility and could be used to detect exogenous biothiols.