Electrochemical reduction of nitrate into ammonia (NO₃^-RR) has attracted great attentions because of the potential application for environmental remediation and energy storage. Despite many pioneering works on electrocatalysts development, the correlation between material properties and NO₃^-RR activity is still not fully understood. Herein, taking layered Ruddlesden-Popper type oxide La₂CuO₄ as a model system, we demonstrate B-site cation doping as an effective approach for modulating NO₃^-RR activities. While Co dopant at Cu site strongly enhanced the NO₃^-RR activity and NH₃ selectivity, Zn and Ni dopants lead to slightly enhanced and suppressed NO₃^-RR performance, respectively. The distinct reaction kinetics of NO₃^-RR on La₂Cu_0.8M_0.2O₄ with different dopants are further revealed by in-situ Fourier transform infrared spectroscopy measurement. Synchrotron-based X-ray absorption spectroscopy showed that B-site doping can effectively regulate metal-oxygen hybridization, leading to strongly tuned surface adsorption characteristics and NO₃^-RR activity. The results of this work can guide the design of highly active (electro)catalysts for environmental and energy devices.