<p>Defect engineering can create various vacancy configurations in catalysts by finely tuning the local electronic and geometric structures of the active sites. However, achieving precise control and identification of these defects remains a significant challenge, and the origin of vacancy configurations in catalysts, especially clustered or associated ones, remains largely unknown. Herein, we successfully achieve the controllable fabrication and quantitative identification of triple O-Ti-O vacancy associate (V_OV_TiV_O) in nanosized Ni-doped TiO₂. Experimental and theoretical analyses demonstrate that terminal hydroxyls adsorbed at unsaturated cationic sites play an essential role in boosting V_OV_TiV_O formation, which enhances H₂O dissociation and facilitates dissociative OH* deprotonation for defect site regeneration. In contrast, a single V_O can be easily saturated by dissociative bridging hydroxyl accumulation, leading to a gradual decrease in the number of active sites. The essential role of V_OV_TiV_O in the Ni-doped TiO₂ is evidenced by its comparable catalytic performance in the hydrogen evolution reaction and hydrodechlorination reactions. Our work highlights the importance of engineering vacancy-associated active sites and presents a notable approach for designing highly active and selective catalysts for efficient H₂O-involved reactions.</p>