The construction of definitive correlation between structure and catalytic properties for electrochemical artificial N2 fixation is still challenging yet significative. Herein, we have explored the coordinative unsaturation (Fe-N2) in isolated Fe atoms with nitrogen vacancies (VN, which are located at the opposite coordination sites of the Fe atom) on interconnected carbon for electrocatalytic ammonia synthesis. Benefiting from dual active centers (Fe-N2 and VN) and the spin density on both of them, the novel catalyst exhibits a synergistic improvement of catalytic activity, selectivity, and stability. This work guides the design of the catalyst theoretically and synergistically and combines the advantages of Fe-N2 with VN structure creation, defect and spin-state engineering introduction toward the concurrent fulfillment of effective N2 adsorption and activation. We believe our strategy will incite more investigation into designing high-performance catalysts with well-defined coordinative unsaturation single-atom active sites for ambient ammonia synthesis and other energy conversion system.