Nanozymes are nanomaterials with natural enzyme characteristics, which are expected to be potential substitutes of traditional enzymes. In this study, the 1Al/MIL-100(Fe) nanozymes with excellent peroxidase-like activity were synthesized by the ultrathin modification (one-cycle Al 2 O 3 ) strategy of atomic layer deposition, whose peroxidase-like activity is almost doubled compared with MIL-100(Fe) nanozymes. Investigation of the catalytic mechanism indicates that the increased amount of hydroxyl radical is responsible for the much-enhanced peroxidase-like activity of 1Al/MIL-100(Fe) nanozymes. Furthermore, the catalytic effect of the designed nanozymes is inhibited by glutathione through consuming the oxidized 3,3′,5,5′-tetramethylbenzidine in the reaction system. The 1Al/MIL-100(Fe) nanozymes also achieve the sensitive and selective detection of glutathione, which have an excellent linear response to glutathione concentration in the range 0.01−1000 μM with a detection limit of 2.2 nM. The ultrathin modification strategy can be potentially extended to synthesize other high-efficiency nanozyme materials.