Age-related cognitive decline presents a healthcare challenge. While age-related mechanisms are mainly studied in humans, animal models provide key insights. Despite evidence of sex-specific differences in aging and cognition, the impact of age on female rodent behaviour is underexplored. This study investigated age-related behavioural changes in female C57BL/6 mice over 8 months, alongside neurochemical markers amyloid, Tau, and T14, a novel peptide from acetylcholinesterase (AChE) that promotes cell growth/renewal. Behavioural assessments showed an age-dependent decline in nest-building ability and familiar odour discrimination from 10 months. Spatial learning declined at 10 and 13 months, while object recognition memory remained intact from 5 to 13 months of age. Neurochemical analyses revealed a decline in T14 and its receptor α7-AChR during postnatal development and adulthood. However, there was a disparity between AChE expression and its enzymatic activity. T14 levels correlated with phosphorylated tau, but not amyloid, and negatively with nest-building scores, suggesting a role of T14 in age-related behavioural changes. This study highlights early behavioural and molecular indicators of cognitive decline in middle-aged female mice.