Abstract: Fluorometry for the determination of β-D-glucosidase (β-Glu) activity is simple and sensitive, and a new fluorometry method for the determination of β-Glu activity was explored. Carbon Dots (CDs) were prepared by self-exothermic reaction using tea infusion (Gampucha) as carbon source. Based on the inner filter effect (IFE), a novel method for the determination of β-Glu activity was established using CDs as fluorescent probe. The results indicated that the prepared CDs had a graphite-like structure with a particle size of 4.5 nm and were mainly composed of C, O, N atoms. The maximum excitation and emission wavelength of CDs were 320 nm and 385 nm respectively. When p-nitrophenol (p-NP) was added to the CDs solution, the fluorescence intensity of CDs decreased significantly, and there was a good linear relationship between the degree of fluorescence quenching lg(I₀/I) and the concentrations of p-NP. Based on the linear response, a new analytical method for p-NP was developed, and p-NP is the hydrolysis product of p-nitrophenyl β-D-glucopyranoside (p-NPG) in the presence of β-D-glucosidase. Therefore, the activity of β-Glu could be obtained indirectly by quantitative results of p-NP. The detection limit of this method was 0.074 U/L with a linear range of 0.1-20 U/L. It was found that there was a large overlap between the fluorescence emission band (385 nm) of CDs and the absorption peak of p-NP (400 nm), and the fluorescence lifetime of CDs did not change significantly after the addition of p-NP, suggesting that the fluorescence quenching mechanism of CDs by p-NP was IFE. The developed method was simple, sensitive, and rapid, and could be applied in the determination of β-Glu activity in fruits.