In this work, a simple and selective fluorescence sensor approach called 'turn-on-off' for the determination of thiamine (TM) has been developed. As known, the o-phenanthroline (o-phen) has inner fluorescence, though when reacted with zinc ions to form the o-phen-Zn complex the fluorescence intensity was enhanced effectively, while upon addition of TM into the o-phen-Zn complex solution, the intensity of the system was gently quenched, which was termed the 'turn-on-off' probe. Notably, the method possessed highly selective, sensitive determination for TM with a detection limit of 0.25 μmol L and the reduced fluorescence intensity was proportional to the concentration of TM in the range 0.84-80.0 μmol L . Besides, the proposed mechanism was also investigated through exploring the Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR) spectroscopy. Furthermore, this manner was successfully applied into practical samples for TM detection with satisfactory results.