As a important biological species in soil ecosystems, earthworms play a significant role in maintaining the function of soil ecosystems. Chlorpyrifos is one of the most widely used organophosphorus pesticides in China, which is a potential threat to soil organisms represented by earthworms. This study used Amynthas corticis, a dominant species of earthworms commonly found in Yunnan woodlands, as the experimental organism to investigate the changes of neurotransmitters (acetylcholine, dopamine, 5-hydroxytryptamine, glutamic acid) and ion channel characteristic enzymes (Ca2+-Mg2+-ATPase, Ca2+-ATPase) in earthworm brain caused by chlorpyrifos exposure, the changes of earthworm neural behavior such as exercise, learning and memory caused by chlorpyrifos subchronic toxicity, evaluated the changes in earthworm biomarkers using the integrated biomarker response (IBR), and conducted a study of subchronic neurotoxicity of chlorpyrifos exposure in earthworms. The results showed that chlorpyrifos exposure caused abnormal changes in the earthworm neurotransmitters acetylcholine, dopamine, 5-hydroxytryptamine, and glutamate, and that neurological damage by chlorpyrifos led to motor retardation and learning and memory impairment in earthworms. The subchronic neurotoxicity IBR values of 8 and 12 mg·kg-1 chlorpyrifos for earthworms at 28 d exposure were 3.2275 and 4.5246, respectively. The IBR values at 56 d exposure were 3.8848 and 5.8995, respectively, and the subchronic neurotoxicity of chlorpyrifos to earthworms increased with increasing exposure concentration and time of exposure to chlorpyrifos. In summary, our study indicated that although chlorpyrifos subchronic neurotoxicity did not lead to earthworm mortality, it produced neurobehavioral changes in earthworms such as Bradykinesia and memory impairment, which could seriously affect the quality of earthworm survival and was detrimental to earthworm survival and development, leading to a decline in the quality of earthworm populations. Therefore, due to the important role of earthworms in soil ecosystems, a decline in the quality of earthworm populations will exacerbate soil ecosystem risks.