Due to the important physiological role of biothiols, the detection for biothiols is becoming increasingly significant, especially the distinguishing detection of Hcy/Cys. In this paper, a simple long-wavelength dual emission fluorescent probe L was designed and synthesized by two-steps reactions using 4-diethylaminosalicylaldehyde as raw material. Probe L can detect Hcy/Cys in the pH range of 6–9 and the detection limit for Hcy and Cys as low as 2.81 μM and 2.33 μM, while L does not respond to GSH/H2S in THF/PBS (2/8, v/v) solution. Probe L can sense Cys with ultrafast responding within 10 s but can detect Hcy in ~6 min, which Hcy/Cys can be distinguished by the reaction speed. In addition, probe L can individually recognize Hcy with better anti-interference ability and sensitivity and distinguish Hcy from Cys in THF/PBS (2/8, v/v, pH 10). Moreover, probe L has been successfully applied to biological field and imaging Hcy in living cells.