The development of selective butyrylcholinesterase (BChE) inhibitors from natural sources represents a strategic frontier in discovering novel therapeutics for Alzheimer's disease (AD). Employing a rigorous hierarchical virtual screening protocol encompassing high-throughput screening, ensemble molecular docking, and MM/GBSA binding affinity refinement, we systematically interrogated a natural product library containing 60,580 phytochemicals. This computational cascade identified three alkaloid candidates (hirsutine, picrasidine I, and picrasidine T) exhibiting potent BChE inhibition (IC50 < 10 μM) validated through enzymatic assays. Detailed kinetic characterization further established hirsutine and picrasidine I as reversible, mixed-type inhibitors, targeting both the catalytic active site and the peripheral aromatic site (PAS) of BChE. To elucidate the mechanism of interaction, picrasidine I was selected for comprehensive spectroscopic analysis, including molecular fluorescence, time-resolved fluorescence, three-dimensional fluorescence, circular dichroism (CD), and isothermal titration calorimetry (ITC). ITC analysis confirmed a strong affinity between picrasidine I and BChE, yielding a dissociation constant (Kd = 2.97 μM), while fluorescence studies indicated a static quenching mechanism. Molecular dynamics (MD) simulations (50 ns) substantiated the stability of the ligand-protein complex, demonstrated by a backbone RMSD <0.3 nm. Furthermore, ADME predictions suggested favorable blood-brain barrier (BBB) permeability and minimal cytotoxicity in PC-12 neurons. This synergistic computational-experimental paradigm not only successfully identifies new chemotypes as potent BChE inhibitors and unveils their precise molecular mechanism of action, but also provides a practical and highly effective strategy for the rational discovery and development of natural anti-AD agents.