Acetylcholinesterases (AChE) are important enzymes present in vertebrates and insects playing a pivotal role that hydrolyze the neurotransmitter acetylcholine.AChE inhibitors are widely employed as pesticides and drugs and there is mounting evidence that environmental or occupational low dose chronic exposure to AChE inhibitors causes adverse neurological outcomes.The detect AChE inhibitory potential of chemicals and environmental samples, we generated a fluorescent transgenic zebrafish line Tg(hspb11∶eGFP) that expresses a GFP reporter under the control of regulatory elements of hspb11, a small heat shock protein shown to respond to AChE inhibitors.Expression of the transgene mimicked faithfully the pattern of endogenous hspb11 expression and responded also to AChE inhibitors.To examine the specificity of the response to AChE inhibitors, we exposed hspb11 reporter embryos to 14 different compounds, including AChE inhibitors and ion channel modulators.Exposure to known AChE inhibitors induced significant increase in the GFP reporter.This suggests that the Tg(hspb11∶eGFP) zebrafish biosensor provides an in vivo alternative to conventional enzymatic assays for detecting environmental AChE inhibitors, featuring sensitivity, high signal-to-noise ratio, specificity, and potential for increased throughput with its quantitative feature.