Biothiols play a crucial role in maintaining redox balance in organisms, and anomalous levels of biothiols in human organs can lead to various sicknesses and biological disorders. This work developed a novel sensitive fluorescent probe TZ-NBD with double channels for highly efficient recognition of biothiols. TZ-NBD adopts 4-Chloro-7-nitrobenzofurazan (NBD-Cl) as the recognition moiety with simultaneous fluorescence output. By incorporating NBD-Cl with the other fluorophore, benzothiazole dihydrocyclopentachromene derivative (TZ-OH), the dual-channel sensitive fluorescence probe TZ-NBD was built. The existence of Cys/ Hcy could significantly trigger both the green and red fluorescent emissions, which were derived from fluorophores amine-substituted NBD and TZ-OH, respectively. While exposing to GSH, only the red-channel fluorescence signal could be detected, indicating the release of TZ-OH. The phenomena was mainly attributed to the fact that sulfur-substituted NBD has nearly no fluorescence, while amine-substituted NBD shows obvious green fluorescence. In our study, TZ-NBD exhibited dual-channel sensitivity, fast response, and excellent selectivity to biothiols in vitro. Moreover, TZ-NBD was favorably utilized for recognition of biothiols in vivo. We believe that the sensitive fluorescence probe with double channels can afford an alternate approach for monitoring biothiols in organisms and would be useful for studying diseases associated with biothiols.