Objective To investigate the therapeutic effect of pralidoxime combined with atropine and lipid emulsion on a mouse model of organophosphate poisoning. Methods Eighty C57/BL6 strain experimental mice were randomly divided into four groups of 10 each using a digital randomization method. A mouse organophosphate poisoning model was established using organophosphate pesticides. Pralidoxime combined with atropine and lipid emulsion was used for treatment. Results The results showed that compared with the poisoning group, the mice in the treatment group showed significant improvement in physiological indexes such as fasciculation tremor intensity and anti-reflex disappearance time (all P<0.001). In particular, the effect was more pronounced in the combination treatment group with pralidoxime, atropine, and lipid emulsion. In addition, the combination therapy effectively improve the serum levels of acetylcholinesterase (AchE), malondialdehyde (MDA) and S100 calcium binding protein B (S100B) in the poisoning mice (all P<0.001) and promoted the recovery of these biochemical indicators to normal levels after 7 days. In the behavioral test, the number of correct responses in the Y-maze test was significantly higher in the treatment group than in the poisoning group, especially in the mice receiving the combination treatment (all P<0.001). Histopathological examinations showed that the combination of clofosidine and atropine could significantly reduce the brain tissue damage caused by poisoning, and the effect was more significant after the addition of lipid emulsion. Conclusions Pralidoxime combined with atropine can effectively alleviate the symptoms of organophosphate poisoning in mice, and the addition of lipid emulsion can further optimize the therapeutic effect, providing a new treatment strategy for clinical practice.