[Display omitted] • Peroxidase-like activity of Glutathione-Ag nanoparticles (GSH-AgNPs) was investigated. • The GSH-AgNPs nanozyme follows Michaelis-Menten behaviors and shows strong affinity to H 2 O 2. • Vitamin B1 can inhibit the catalytic activity of GSH-AgNPs. • Lable-free colorimetric sensors for the detection ofVitamin B1 was developed with excellent sensitivity and selectivity. A label-free sensing strategy based on the enzyme-mimicking property of Glutathione-Ag nanoparticles (GSH-AgNPs) was demonstrated for colorimetric detection of vitamin B1 (VB1). Firstly, obvious blue color accompanied with an absorption peak at 652 nm was observed due to the high peroxidase-like activity of GSH-AgNPs towards 3,3′,5,5′-tetramethylbenzidine (TMB). Then, in the presence of VB1, the mimetic activity of GSH-AgNPs could be strongly restrained, evidenced as a promiment colorimetric change to colorless, which can be used to achieve the visualization detection VB1. Linear relationship between absorbance response and VB1 concentration from 0 to 0.2 µM were obtained. The detection limit was calculated as low as 40 nM. The inhibition reasons were thoroughly discussed. Considering the advantages of rapid response, easy procedure and high selectivity, the proposed method possesses potential application in environment and biological analysis for VB1 detection. [ABSTRACT FROM AUTHOR]