Electrocatalytic nitrate reduction reaction has attracted increasing attention due to its goal of low carbon emission and environmental protection. Here, we report an efficient NitRR catalyst composed of single Mn sites with atomically dispersed oxygen (O) coordination on bacterial cellulose-converted graphitic carbon (Mn–O–C). Evidence of the atomically dispersed Mn–(O–C₂)₄ moieties embedding in the exposed basal plane of carbon surface is confirmed by X-ray absorption spectroscopy. As a result, the as-synthesized Mn–O–C catalyst exhibits superior NitRR activity with an NH₃ yield rate (R_NH3) of 1476.9 ± 62.6 μg h⁻¹ cm⁻² at − 0.7 V (vs. reversible hydrogen electrode, RHE) and a faradaic efficiency (FE) of 89.0 ± 3.8% at − 0.5 V (vs. RHE) under ambient conditions. Further, when evaluated with a practical flow cell, Mn–O–C shows a high R_NH3 of 3706.7 ± 552.0 μg h⁻¹ cm⁻² at a current density of 100 mA cm⁻², 2.5 times of that in the H cell. The in situ FT-IR and Raman spectroscopic studies combined with theoretical calculations indicate that the Mn–(O–C₂)₄ sites not only effectively inhibit the competitive hydrogen evolution reaction, but also greatly promote the adsorption and activation of nitrate (NO₃⁻), thus boosting both the FE and selectivity of NH₃ over Mn–(O–C₂)₄ sites.