In this work, a new fluorescence probe (DC) with a donor-π-acceptor (D-π-A) structure was designed and synthesized for the detection of three kinds of biothiols (Cys, Hcy and GSH) in live cells and organisms. DC displayed an intense red-emission centered at 625 nm. In the presence of biothiols, nucleophilic addition reaction between C=C double bond of DC and sulfhydryl group (-SH) of biothiols occurred, resulting in obvious fluorescence quenching responses. DC exhibited highly selectivity towards biothiols over other common bioactive species with low detection limits (0.26, 0.43, and 0.44 µM for Cys, Hcy and GSH, respectively). In addition, DC displayed a rapid response to biothiols within 4 min. The applications of DC in biothiols detection and imaging were then successfully demonstrated for the real-time monitoring endogenous and exogenous biothiols in live cells and live animals.