Objective To investigate temporal relationships among neurotransmitter alterations, amyloid-beta (Aβ) deposition, and neuroinflammation in 5 × FAD mice. Methods 5 × FAD and C57BL / 6J (WT) mice at 1. 5 months (early pathological stage), 3 months (mid-pathological stage), and 6 months (late pathological stage) were examined to establish a time-window model of Alzheimer’s disease(AD) progression. Each age and genotype group included 8 mice. Behavioral phenotyping was assessed using the Y-maze, novel object recognition, and open field tests. Immunofluorescence was used to detect Aβ expression and glial cell activation in the hippocampus and prefrontal cortex (PFC). HE staining was performed to observe hippocampal neuronal morphology. Neurotransmitter levels in the hippocampus and PFC were quantified via high-performance liquid chromatography. Results At 1. 5 months, the hippocampal GABA level was lower in AD model group mice than in WT group mice. At 3 months,significant changes in several neurotransmitters, including acetylcholine, glutamate, serotonin, and 5- hydroxyindoleacetic acid (5-HIAA), were detected in the hippocampus of AD model group mice compared with WT group mice. At this stage, minor Aβ deposition and microglial activation were present in both the hippocampus and PFC. By 6 months, neurotransmitter levels further declined in the hippocampus and PFC of AD model group mice,accompanied by extensive Aβ deposition, pronounced hippocampal neuronal damage, and substantial glial cell activation. Cognitive impairments were also observed. Conclusions Neurotransmitter dysregulation in 5 × FAD mice emerged by 3 months of age and worsened with disease progression. These alterations were closely associated with Aβ deposition, neuroinflammation, and neuronal injury.