Electrocatalytic ammonia synthesis under mild conditions is an attractive and challenging process in the earth's nitrogen cycle, which requires efficient and stable catalysts to reduce the overpotential. The N2 activation and reduction overpotential of different Ti3C2O2-supported transition metal (TM) (Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Mo, Ru, Rh, Pd, Ag, Cd, and Au) single-atom catalysts have been analyzed in terms of the Gibbs free energies calculated using the density functional theory (DFT). The end-on N2 adsorption was more energetically favorable, and the negative free energies represented good N2 activation performance, especially in the presence Fe/Ti3C2O2 (-0.75 eV). The overpotentials of Fe/Ti3C2O2, Co/Ti3C2O2, Ru/Ti3C2O2, and Rh/Ti3C2O2 were 0.92, 0.89, 1.16, and 0.84 eV, respectively. The potential required for ammonia synthesis was different for different TMs and ranged from 0.68 to 2.33 eV. Two possible potential-limiting steps may be involved in the process:(i) hydrogenation of N2 to *NNH and (ii) hydrogenation of *NH2 to ammonia. These catalysts can change the reaction pathway and avoid the traditional N-N bond-breaking barrier. It also simplifies the understanding of the relationship between the Gibbs free energy and overpotential, which is a significant factor in the rational designing and large-scale screening of catalysts for the electrocatalytic ammonia synthesis.