Objective To explore the temporal relationship between the dynamic changes of neurotransmitters, amyloid-beta (Aβ) deposition and neuroinflammation in 5×FAD mice. Methods 5×FAD and WT mice at 1.5 months (early pathological stage), 3 months (mid-pathological stage), and 6 months (late pathological stage) were selected to establish a time window model of Alzheimer"s disease (AD) progression. Behavioral phenotyping was quantified using the Y-maze, novel object recognition and open field tests. Immunofluorescence was used to detect Aβ expression and glial cell activation levels in the hippocampus and prefrontal cortex (PFC). Hematoxylin and eosin (HE) staining was used to observe hippocampal neuronal morphology. Additionally, neurotransmitter levels in the hippocampus and PFC were quantified using high-performance liquid chromatography (HPLC). Results The study found that at 1.5 months of age, the GABA level in the hippocampus of 5×FAD mice was lower than that of WT mice. At 3 months of age, significant changes were observed in the levels of several neurotransmitters, including acetylcholine, glutamate, serotonin, and 5-hydroxyindoleacetic acid, in the hippocampus of 5×FAD mice compared to WT mice. At this time, there was minor Aβ deposition in both the hippocampus and PFC, along with activated microglia. At 6 months of age, neurotransmitter levels continued to decline in the hippocampus and PFC of 5×FAD mice, with abundant Aβ deposition, significant hippocampal neuronal damage and increased activation of glial cells. Cognitive behavioral impairments also appeared. Conclusion Neurotransmitter dysregulation in 5×FAD mice was observed as early as 3 months of age and progressively worsened with disease progression. This change was closely associated with Aβ deposition, neuroinflammation, and neuronal damage.