As a non-invasive therapeutic approach, 40 Hz light flicker stimulation is deemed to hold considerable promise in the treatment of Alzheimer's disease (AD). However, whether long-term effects of 40 Hz light flicker stimulation can improve working memory and its related mechanisms remain to be further explored. In this study, 10 adult Wistar rats were randomly divided into AD light stimulation group and AD group, with 5 rats in each group. AD rat models were prepared for the AD light flicker stimulation group and AD group, and the AD light flicker stimulation group was subjected to 40 Hz light flicker for 7 days. Subsequently, the spatial working memory function of each group of rats was detected based on the T-maze task, and the potential neural mechanism of the improvement of working memory performance by 40 Hz light flicker stimulation was further explored from the perspective of the intensity and synchronism of neural activity in the hippocampus (HPC) and the prefrontal cortex (mPFC) related to working memory. The results showed that the long-term effect of 40 Hz light flicker improved the spatial working memory function of AD rats, and enhanced the synchronization of neural activity between HPC-mPFC, and enhanced the level of information transmission between brain regions mediated by θ and low frequency gamma oscillations. This study provides a theoretical basis for the use of 40 Hz light flicker stimulation as a potential method to prevent or treat AD disease.