A chemluminescence (CL) biosensor responding to acetylcholine (ACh) and choline (Ch) was prepared by covalently coupling acetylcholinesterase (AChE) and choline oxidase (ChO) to the controlled pore glass as a molecular recognition element, using electrostatically immobilizing luminol and copper ion exchange resin as a transduction element in a flow injection system. The analystes were injected into a continuous stream of simple medium flowing through a sequence of enzyme reactors containing AChE and ChO to produce hydogen peroxide. The luminol and Cu^2^+ were eluted by Na~2SO~4 from the ion exchange resin column, then reacted with hydrogen peroxide to produce CL. The linear range for both the ACh and Ch was 1～1000 pmol. The detection limit was 500 fmol. A complete analysis can be finished within 2min. The sensor was stable for 6 months and has been applied successfully to the analysis of ACh and Ch in human serum and rat brain.

Key words: BIOSENSORS, CHEMILUMINESSENCE, CHOLINE, ACETYLCHOLINE, CHOLINESTERASE, LUMINOL, OXIDASE