Acetylcholinesterase (AChE) inhibitors are mainly used in the treatment of Alzheimer’s disease (AD).  The inhibitory effect of icariin on the activity of AChE was investigated by inhibition kinetics.  The binding interaction and binding sites between icariin and AChE were also studied by using fluorimetry and molecular docking, respectively.  The results showed that icariin could potently inhibit the activity of AChE, the IC₅₀ value was determined to be 3.50×10⁻⁸ mol·L⁻¹, and the determined IC₅₀ value to tacrine was 0.75×10⁻⁸ mol·L⁻¹.  Kinetic analyses showed that icariin is a reversible and mixed type AChE inhibitor.  The inhibition constants K_I and K_IS were determined to be 2.67×10⁻⁸ and 4.43×10⁻⁸ mol·L⁻¹, respectively.  Icariin binds selectively to the AChE peripheral anionic site via hydrogen bonds and Van der Waals forces.