We report herein a new colorimetric assay method for acetylcholinesterase (AChE) activity and its inhibitor screening by making use of the following facts: (1) the aggregation of gold nanoparticles (Au-NPs) results in the red-shift of the plasmon absorption due to interparticle plasmon interactions and (2) AChE can catalyze the hydrolysis of acetylthiocholine into thiocholine which can induce the aggregation of Au-NPs. With this convenient method, the activity of AChE with a concentration as low as 0.6 mU/mL can be assayed. Moreover, this assay method is also useful for screening inhibitors of AChE. Given its simplicity and easy-operation, this method may extend to high-throughput screening of AChE inhibitors and relevant drug discovery.