Trichlorfon is an organophosphorus pesticide widely used in aquaculture and has potential neurotoxicity, but the underlying mechanism remains unclear. In the present study, zebrafish embryos were exposed to trichlorfon at concentrations (0, 0.1, 2 and 5 mg/L) used in aquaculture from 2 to 144 h post fertilization. Trichlorfon exposure reduced the survival rate, hatching rate, heartbeat and body length and increased the malformation rate of zebrafish larvae. The locomotor activity of larvae was significantly reduced. The results of molecular docking revealed that trichlorfon could bind to acetylcholinesterase (AChE). Furthermore, trichlorfon significantly inhibited AChE activity, accompanied by decreased acetylcholine, dopamine and serotonin content in larvae. The transcription patterns of genes related to acetylcholine (e.g.,ache,chrna7,chata,hactandvacht), dopamine (e.g.,drd4aanddrd4b) and serotonin systems (e.g.,tph1,tph2,tphr,serta,sertb,htrlaaandhtrlab) were consistent with the changes in acetylcholine, dopamine, serotonin content and AChE activity. The genes related to the central nervous system (CNS) (e.g.,a1-tubulin,mbp,syn2a,shhaandgap-43) were downregulated. Our results indicate that the developmental neurotoxicity of trichlorfon might be attributed to disorders of cholinergic, dopaminergic and serotonergic signaling and the development of the CNS.