β-Amyloid (Aβ) plaque, representing the progressive accumulation of the protein that mainly consists of Aβ, is one of the prominent pathological hallmarks of Alzheimer’s disease (AD). Label-free imaging of Aβ plaques holds the potential to be a histological examination tool for diagnosing AD. We applied label-free multiphoton microscopy to identify extracellular Aβ plaque as well as intracellular Aβ accumulation for the first time from AD mouse models. We showed that a two-photon-excited fluorescence signal is a sensitive optical marker for revealing the spatial–temporal progression and the surrounding morphological changes of Aβ deposition, which demonstrated that both extracellular and intracellular Aβ accumulations play an important role in the progression of AD. Moreover, combined with a custom-developed image-processing program, we established a rapid method to visualize different degrees of Aβ deposition by color coding. These results provide an approach for investigating pathophysiology of AD that can complement traditional biomedical procedures.