Calcium ions are important messengers in the nervous system, which produce various cellular signals and play an important role in the regulation of neuronal excitability. Calcium ions in the clear cell area perform highly specific functions. Calcium signals indirectly reflect the activity of neurons, calcium signal detection in neurons is especially important to study cerebral cortex functions. Two-photon calcium imaging in the cerebral cortex has a unique advantage by revealing real-time in vivo activity of cortical neurons at the single cell resolution. This article reviews the application of two-photon calcium imaging in the study of cortical functions in non-human primates.