The efficient conversion of solar energy into chemical fuels, using solar-energy-generated electricity to drive electrocatalytic (EC) or photoelectrocatalytic (PEC) water splitting, is still a grand challenge in sustainable energy due to the sluggish oxygen evolution reaction (OER). Two-dimensional (2D) semiconductors are promising catalysts in both cases, yet their performance is governed by a complex and heterogeneous landscape of atomic-scale defects that remains convoluted. Here, a multi-resolved imaging strategy, combining in situ electrochemiluminescence (ECL), photoinduced ECL, and photoluminescence imaging modes with ex situ scanning transmission electron microscopy (STEM) analysis, was employed to provide comprehensive insights into the dynamic heterogeneous relationship between catalytic activity, carrier behaviors and active sites on 2D semiconducting materials. By investigating pristine and annealed monolayer MoS₂, we establish a direct link between the distribution of specific defect types and their unique influence on catalytic activity and carrier behavior. We’ve identified molybdenum vacancies (V_Mo) as bifunctional active sites for both EC and PEC OER, while sulfur vacancies (V_S), while not primary catalytic centers, uniquely enhance PEC efficiency. This work provides a powerful methodology for elucidating complex, dynamic catalytic mechanisms, paving the way for the defect-level engineering of high-performance 2D materials-based catalysts.