The improvement of analytical accuracy and S/N (signal to noise ratio) based on conventional homogeneous fluorometry was always limited because of high background noise from complicated sample ingredients, buffer solution and lysates. Herein, a self-assembled Mn-doped ZnSe quantum dot–methyl viologen nanohybrid as an OFF–ON fluorescent probe is presented for the time-resolved fluorescence detection of tiopronin, showing high S/N and selectivity with a low detection limit of 0.1 μmol L⁻¹ and a linear response ranging from 4 to 160 μM. We anticipate that this probe will have promising applications for sensitive biosensing and drug analysis.