Alzheimer’s disease is one of the most common causes of dementia. Acetylcholinesterase has been considered as the main therapeutic target in the treatment of Alzheimer’s disease. Natural products are the richest source of lead compounds for the discovery and development of new drugs. In the present contribution, the hierarchical biology–oriented method was applied to discover the active gradients of F. pseudalliacea root as AChE inhibitors. The Kamonolol acetate was extracted and identified from the n-hexane extract of F. pseudalliacea root. The Kamonolol acetate inhibited the AChE with IC₅₀: 63.9 μM. Molecular modeling studies showed that Kamonolol acetate interacted with CAS and PAS of AChE active site. Kinetics in accompany with molecular modeling studies demonstrated that Kamonolol acetate inhibited AChE in the mixed-type model. The results indicated that Kamonolol acetate could be considered as a valuable lead compound in the design of AChE inhibitors.