Calcium is an important messenger in the mammalian nerve cells which mediates a variety of intracellular signal transduction pathways and plays critical roles in regulating the neuronal functions. Calcium signaling exerts its highly specific function in a defined sub-region of the cell, especially in the visual cortex of the brain. Detection of calcium signals in neurons is particularly important for the studying of neuronal function. The two-photon microscope has a unique advantage in the detection of calcium signal in the superficial cortex. In this paper, the application of two-photon in the in vivo detection of the visual cortical Ⅱ/Ⅲ layer of model animals are reviewed.