Here, the MgAl layered double hydroxide (LDH) supports and relevant metal on LDH catalysts (i.e., Ni, Co, and Ru) were prepared and investigated under non thermal plasma (NTP) conditions to probe the role of surface oxygen vacancy (OV) in NTP-assisted ammonia (NH3) synthesis. The findings show that OV on the LDH carrier is highly beneficial to NH3 formation under NTP conditions, and concentration of OV on the LDH can be regulated by the post-synthesis calcination and hydrogen plasma etching. Additionally, loading of active metal species on the LDH could promote the NH3 synthesis further due to presence of multiple reaction pathways and the synergy between the surface OV and metal sites in such NTP-catalytic systems. As the result, the catalysts developed by this work showed high ammonia synthesis rates of 4.42−4.52 mmol g−1 h−1 and energy efficiencies of 1.67−1.71 gNH3 kWh−1, respectively. The findings of the work pave the way for the rational design and optimization of highly efficient catalysts with dual active sites for intensifying the NTP-catalytic ammonia synthesis.