A fluorescence assay for the detection of 4-nitrophenol (4-NP), α-glucosidase (α-Glu) activity and α-Glu inhibitors (AGIs) is developed based on the inner filter effect (IFE), a flexible and simple signal transfer strategy. In this assay, silicon nanoparticles (Si NPs) synthesized under mild and easily accessible conditions are employed as fluorescent indicators. 4-NP efficaciously quenches the fluorescence of Si NPs through the IFE at a very rapid rate, thus achieving 4-NP detection in a mix-to-read manner, which is suitable for on-site detection. The quenching mechanism has been comprehensively studied and confirmed. More significantly, based on the fact that 4-NP can be generated through α-Glu-catalyzed hydrolysis of 4-nitrophenyl-α-D-glucopyranoside (NPG), the fluorescence detection of α-Glu activity is legitimately achieved by employing NPG as the substrate. The linear ranges for 4-NP and α-Glu activity detection are 0.5–60 μM and 0.5–60 mU mL⁻¹ with low detection limits of 0.074 μM and 0.094 mU mL⁻¹, respectively. This method not only can preciously assay targets in real samples, but is also capable of screening AGIs as drugs as well as assessing their inhibition efficiency.