Thiols and primary aliphatic amines (PAA) are ubiquitous and extremely important species in biological system. They have performed significant interplaying roles in complex biological events. A single fluorescent probe differentiating both thiol and PAA can contribute to understanding the intrinsic inter-relationship of thiols and PAA in biological processes. Herein, we rationally constructed the first fluorescent probe that can respond to thiols and PAA in different fluorescence channels. The probe exhibited high selectivity and sensitivity to thiols and PAA. In addition, it displayed sequential sensing ability when the thiols and PAA were coexisted. The application experiment indicated that the probe can be used for sensing thiols and PAA in human blood serum. Moreover, the fluorescence imaging of endogenous thiols and PAA as well as antihypertensive drugs captopril and amlodipine in living cells were successfully conducted.