A method for creating an immobilized capillary acetylcholinesterase (AChE) reactor based on layer-by-layer assembly (LBL) for inhibitor screening is described.The unique capillary AChE reactor was easily prepared by the instrument in three steps: first, a 0.5 cm long plug of the solution of cationic polyelectrolyte polydiallyldimethylammonium (PDDA) was injected into the capillary to produce a positively charged coating on the surface of the capillary; subsequently, the enzyme solution with the same plug length was injected into the capillary and incubated for 10 min to immobilize the enzyme on the capillary wall via electrostatic interaction; third, PDDA solution with the same plug length was injected again into the capillary to cover the immobilized enzyme by forming PDDA-AChE-PDDA sandwich-like structure.The enzyme reactor can be easily renewed after removing the immobilized enzyme by flushing the column with 1 M NaC1 solution.Activity of the immobilized enzyme can be assayed simply by carrying out an electrophoretic separation, i.e.the substrate solution was injected and incubated for a short time, followed by applying a voltage to separate the product fiom the unreacted substrate.