Single-atom catalysts (SACs), specifically iron single atoms dispersed on nitrogen-doped carbon (Fe-NC), have shown promising potential in the electrocatalytic reduction of nitrate to ammonia (NitRR), but there is a lack of understanding of their intrinsic activity. The conventional measurements often overlook the intrinsic performance of SACs, leading to significant underestimation. This study presents an in situ electrochemical probing protocol, using two poisoning molecules (SCN- and NO2-), to characterize the reactivity of Fe sites in Fe-NC SACs for NitRR. The technique aids in quantifying the yield rate of ammonia on Fe sites and the active site number. The findings reveal the intrinsic turnover frequency (TOF) based on the number and ammonia yield rate of Fe sites, challenging the current understanding of SACs' inherent performances. This unique approach holds considerable potential for determining the intrinsic activity of other SACs in complex reactions, opening new avenues for the exploration of electrocatalytic processes.