Hydrogen sulfide (H2S) and biothiols play close interrelated roles in complex physiological and pathological events. Differentiating H2S and biothiols is highly significant to dissect their interplaying roles in biological system. Herein, a bifunctional fluorescent probe, OY, that could differentiate H2S and biothiols has been rationally constructed. Probe OY possessed two different recognition sites for detecting H2S and biothiols respectively, so as to avoid two analytes competing for one recognition site. Moreover, optical studies revealed that probe OY was able to selectively detect H2S and biothiols in red and blue fluorescence channels correspondingly. Furthermore, the sensing reaction of probe OY to H2S is much faster than that to biothiols. Therefore, probe OY was capable of detecting H2S and biothiols in two-step cascade mode in distinct fluorescence channels, which will dramatically mitigate potential crosstalk detection between H2S and biothiols. Practical application demonstrated that probe OY could be utilized for differentiating H2S and biothiols in living cells. Also, the variations of H2S and biothiols concentrations during non-enzymatical production of H2S were monitored. In addition, probe OY was used to track dynamic levels of H2S and biothiols in crop plant tissues which cultivated under different concentration of atmospheric H2S.