The use of some organophosphate insecticides is restricted or even banned in paddy fields due to their high toxicity to aquatic organisms. The aim of this study is to elucidate the main pathways and target organs of organophosphate insecticide toxicity to fish exposed via different routes by integrating histopathological and biochemical techniques. Using malathion as the model drug, when the dosage is 20-60 mg/L, the toxicity of whole body and head immersion drugs to zebrafish is much higher than that of trunk immersion drugs. A dose of 21.06-190.44 mg/kg of malathion feed was fed to adult zebrafish. Although the dosage was already high, no obvious toxicity was observed. Therefore, we believe that the drug mainly enters the fish body through the gills. When exposed to a drug solution of 20 mg/L and 60 mg/L, the fish showed significant neurological behavioral abnormalities, and the pathological damage to key organs and brain tissue was the most severe, showing obvious vacuolization and the highest residual amount (8.72-47.78 mg/L). The activity of acetylcholinesterase was the most inhibited (54.69-74.68%). Therefore, brain tissue is the key toxic target organ of malathion in fish. In addition, we compared the bioaccumulation effects of different water-soluble organophosphorus insecticides in fish and their toxic effects. We found that the higher the water solubility of organophosphorus insecticides, the lower their toxicity to fish.