Portable and sensitive mixed-potential type solid state electrolyte (MPSE) gas sensors can detect exhaled biomarkers in a noninvasive and inexpensive way, which is significant for convenient disease diagnosis and saving medical resources. However, high working temperature is still one of the main bottlenecks for hindering MPSE gas sensors’ applications in disease diagnosis. Here, we, for the first time, developed and fabricated new room-temperature MPSE gas sensors utilizing K2Fe4O7 electrolyte and Ni/Fe−MOF (Ni/Fe clusters are coordinated with 1,4-H2BDC) sensing electrodes (SEs) for the detection of ppb-level NO. Among different MOF SEs, the sensor attached with the Ni−MOF SE presents the highest NO sensitivities. This is attributed to a reducing oxygen reduction reaction activity and enhancing NO electrochemical catalytic reaction activity, verified by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) tests. In addition, the presented sensor also shows a low detection limit (20 ppb), fast response/recovery characteristic (17 s/6 s to 50 ppb NO), excellent selectivity, acceptable repeatability, and long-term stability of 34 days to NO at 25 °C and 60%RH. Simultaneously, the mechanism of humidity effect on the sensing performance was investigated by EIS and CV tests. Our work provides new insight into the development of room-temperature solid-state electrolyte gas sensors based on the mixed-potential mechanism and enlarges the potential application domain