We demonstrate a carbon nanotube based high-resolution biosensor for acetylcholine sensing. Carbon nanotubes are deposited on a silicon wafer in a repeated fashion with layer-by-layer nano self-assembly technique. With nano-assembled acetylcholinesterase molecules on the surface, the carbon nanotube biosensor is capable of detecting acetylcholine at an ultra-low concentration of 100 pM. The sensitivity of the acetylcholine sensor is measured as 7.2 µA/decade. The real-time measurement shows the response time of the biosensor is approximately 6 sec. Both the carbon nanotube film and the acetylcholinesterase film are crucial in the sensing process. Due to its high resolution, fast response, small size, and low cost, the carbon nanotube biosensor has tremendous potential for applications in medical research and clinical diagnosis.