Fluoxetine (FLX) has broad neurobiological functions and neuroprotective effects; however, the preventive effects of FLX on cognitive impairments in Alzheimer's disease (AD) have not been reported. Here, we studied whether adolescent administration of fluoxetine can prevent memory deficits in AD transgenic mice that harbour PS1m146v, APPswe and TauP301L mutations (3 × TgAD). FLX was applied through peritoneal injection to the mice at postnatal day 35 (p35) for 15 consecutive days, and the effects of FLX were observed at 6-month. We found that adolescent administration of FLX improved learning and memory abilities in 6-month-old 3 × TgAD mice. FLX exposure also increased the sizes of the hippocampal CA1, dentate gyrus (DG) and extensive cortex regions, with increased numbers of neurons and higher dendritic spine density. Meanwhile, the synaptic plasticity of neurons in the hippocampus was remodelled, and the expression levels of synaptic-related proteins were increased along with activation of the cyclic AMP response element-binding (CREB) protein/brain-derived neurotrophic factor (BDNF) signalling pathway. Finally, we found that FLX effectively prevented the increase of beta-amyloid (Aβ) levels. These data suggest that adolescent administration of the antidepressant drug FLX can efficiently preserve cognitive functions and improve pathologies in 3×Tg AD mice.