Conventional ammonia production consumes significant energy and causes enormous carbon dioxide (CO2) emissions globally. To lower energy consumption and mitigate CO2 emissions, a facile, environmentally friendly, and cost-effective one-pot method for the synthesis of a ruthenium-based nitrogen reduction nanocatalyst has been developed using reduced graphene oxide (rGO) as a matrix. The nanocatalyst synthesis was based on a single-step simultaneous reduction of RuCl3 into ruthenium-based nanoparticles (Ru-based NPs) and graphene oxide (GO) into rGO using glucose as the reducing agent and stabilizer. The obtained ruthenium-based nanocatalyst with rGO as a matrix (Runano-based/rGO) has shown much higher catalytic activity at lower temperatures and pressures for ammonia synthesis than conventional iron catalysts. The rGO worked as a promising promoter for the electrochemical synthesis of ammonia due to its excellent electrical and thermal conductivity. The developed Runano-based/rGO nanocatalyst was characterized using transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), ultraviolet–visible (UV–vis) absorption spectroscopy, X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS), inductively coupled plasma mass spectrometry (ICP-MS), and X-ray photoelectron spectroscopy (XPS). The results demonstrated that the size of the Ru-based NPs on the surface of rGO was 1.9 ± 0.2 nm and the ruthenium content was 25.03 wt %. Bulk electrolysis measurements were conducted on thin-layer electrodes at various cathodic potentials in a N2-saturated 0.1 M H2SO4 electrolyte at room temperature. From the chronoamperometric measurements, the maximum faradic efficiency (F.E.) of 2.1% for ammonia production on the nanostructured Runano-based/rGO electrocatalyst was achieved at a potential of −0.20 V vs reversible hydrogen electrode (RHE). This electrocatalyst has attained a superior ammonia production rate of 9.14 μg·h–1·mgcat. –1. The results demonstrate the feasibility of reducing N2 into ammonia under ambient conditions and warrant further exploration of the nanostructured Runano-based/rGO for electrochemical ammonia synthesis.