Biological thiols (biothiols), such as glutathione (GSH), cysteine (Cys) and homocysteine (Hcy), play a vital role in the process of reversible redox reactions in physiological systems. In this work, flow cytometry-based fluorescent sensor is for the first time developed for the detection of biothiols in a fluorescence “turn on” manner. The probe which we name “Polystyrene/Quantum Dots/Gold Nanoparticles” or (PS/QDs/Au) is constructed by immobilizing QDs onto the surface of PS microbeads to obtain fluorescent microbeads. The probe (PS/QDs/Au) is constructed by immobilizing QDs onto the surface of PS microbeads to obtain fluorescent microbeads, followed by gold NPs absorption through electrostatic interaction to quench their fluorescence. In the presence of biothiols, the fluorescence of our probe can be restored in less than 5 min, and the detection limits for GSH, Cys and Hcy are 0.5 μM, 0.1 μM and 0.3 μM, respectively. Most importantly, the fluorescence signal of each of our probe microbeads can be collected individually by flow cytometry, realizing single microbead-based biothiols detection for the first time. Moreover, the probe is successfully applied to imaging of intracellular biothiols in A549 cells, demonstrating its potential in biological application.