In this paper, a new low dose fluorescent probe with excellent sensing properties for detection of biothiols (GSH, Cys and Hcy) was developed. This probe shows rapid response, high selectivity and sensitivity for biothiols with dual colorimetric and fluorescent turn-on signal changes. The detection limit of this probe for biothiols was found to be <10 nM based on the fluorescent detection. More importantly, this probe can be used to image biothiols effectively in living cells even with a low dose of 0.2 μM.