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The insightful comprehension of in situ catalytic dynamics at individual structural defects of two-dimensional (2D) layered material, which is crucial for the design of high-performance catalysts via defect engineering, is still missing. Here, we resolved single-molecule trajectories resulted from photocatalytic activities at individual structural features (i.e., basal plane, edge, wrinkle, and vacancy) in 2D layered indium selenide (InSe) in situ to quantitatively reveal heterogeneous photocatalytic dynamics and surface diffusion behaviors. The highest catalytic activity was found at vacancy in a four-layer InSe, up to ~30× higher than that on the basal plane. Moreover, lower adsorption strength of reactant and slower dissociation/diffusion rates of product were found at more photocatalytic active defects. These distinct dynamic properties are determined by lattice structures/electronic energy levels of defects and layer thickness …