The aim of this study was to investigate the in vitro inhibitory activity of aloe-emodin on cholinesterase and the in vivo effect of aloe-emodin on cholinesterase activity in the brain, extent of spatial and object recognition, as well as learning and memory ability in a mouse model of Alzheimer's disease (AD). In vitro cholinesterase inhibitory activity was measured by the Ellman’s colorimetric method, while in vivo effect was assessed by using a mouse model of scopolamine-induced dysmnesia. Mice were randomly divided into four groups: normal group, model group, huperzine A positive-control group, and aloe-emodin group. The learning and memory ability was measured by using the Morris water-maze. The ameliorating effect due to aloe-emodin was measured by using behavioral experimentation (water-maze task) and brain cholinesterase activity as indicators. Aloe-emodin was found to effectively inhibit cholinesterase activity in vitro (IC50 = 18.37 μg/mL). Compared with the model group, aloe-emodin showed a significant decrease in escape latency, an increase in swimming time in the target quadrant, a reduction in brain cholinesterase activity, and an improvement in the learning and memory ability of the animal model. Thus, aloe-emodin was shown to ameliorate learning and memory deficit induced by scopolamine in mice, and the underlying mechanism may be related to inhibition of cholinesterase activity.