The enzyme α-L-fucosidase (AFu) plays an important role in the diagnosis of hepatocellular carcinoma (HCC) and fucosidosis. In this paper, a simple, sensitive and precise method based upon measuring the fluorescence quenching of CdTe semiconductor quantum dots (QDs) was developed for detecting the enzymatic activity of AFu. The detection limit of AFu was 0.01 U/L (n = 3) and the linear relationship was 0.01-4 U/L. The selectivity experiment indicated excellent selectivity for AFu over a number of interfering species. We have also studied the detection mechanism of AFu by X-ray photoelectron spectroscopy (XPS) and found that the quenching effect was caused by the oxidation of tellurium by 2-chloro-4-nitrophenol (2-CNP) which produced in AFu catalytic reaction. Moreover, the AFu sensor based on QDs was used satisfactorily for the assessment of AFu activity in serum samples. It will most probably be applicable in assembling diagnostic microdevice to realize the rapid clinic analysis of AFu.