A sensitive nanocomplex probe prepared from fluorescent polydopamine nanoparticles (F-PDA) and cobalt oxyhydroxide (CoOOH) nanosheets was established for the determination of α-glucosidase activity. In this detection system, the fluorescence of F-PDA was firstly quenched by CoOOH nanosheets based on fluorescence resonance energy transfer (FRET). Subsequently, ascorbic acid was produced from 2-O-α-d-glucopyranosyl-l-ascorbic acid which was selectively hydrolyzed by α-glucosidase. CoOOH was reduced to Co2+ by the released ascorbic acid, which resulted in the recovery of F-PDA nanoparticles fluorescence. In consequence, α-glucosidase activity was determined by the fluorescence recovery degree of the F-PDA nanoparticle. This fluorescent method showed a good linear relationship with the activity of α-glucosidase from 2 to 80 U L-1 and low detection limit of 1.65 U L-1 (S/N = 3). This fluorescence probe with high selectivity and sensitivity demonstrated a remarkable applicability in human serum samples and provided an alternative for α-glucosidase inhibitors screening in the discovery of anti-diabetes drugs.