Alpha-glucosidase (α-Glu) is a hydrolase that promotes the rise of glucose in the blood during the digestion process, and it plays a crucial role in regulating glucose levels within a normal range. Herein, we developed an ultrasensitive colorimetric platform by preparing a Cu–N–C single-atom nanozyme (Cu–N–C SAzyme) with a potent oxidase-like effect on accurately detection of α-Glu and efficient screening of its potential inhibitors. This artificial nanoenzyme was able to catalyze the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxide (oxTMB) without the assistance of H₂O₂. In addition, with the participation of α-Glu, L-ascorbic acid-2-O-α-D-glucopyranose (AAG) can be catalyzed to produce ascorbic acid (AA), which significantly reduces the oxidation, resulting in a noticeable fading of the blue color. Therefore, we designed a straightforward colorimetric test for α-Glu activity, achieving detection limits as low as 0.09 U/L and a linear response from 0.25 to 8 U/L. Most importantly, this platform has been effectively applied to measure α-Glu in real samples.