Abstract: This paper reported the manufacture method, working principle and operating method of double-barreled NH4+ selective microelectrode. An apparent difference of NH4+ selective microelectrode from other microelectrodes (such as H+ and NO3- microelectrode) is the interference of K+. These microelectrodes showed a typical log linear response to NH4+ from 0.01–100 mmol/L in the presence of 72mmol/L K+, with a slope of 48 ~58 mV. The detect limit was below 10-3mmol/L. The results showed that the microelectrode was more sensitive and selective to NH4+ and could be used on the NH4+ measurement. Intracellular measurement of NH4+ activity in leaf cells of rice using the double-barreled NH4+ selective microelectrode showed that NH4+ activity fell into two main populations, one was in the cytoplasm with the NH4+concentrations of 2.58–9.37 mmol/L and the other one was in the vacuole with the NH4+concentrations of 11.36–25.20 mmol/L. Tissue NH4+ of rice leaf mainly comes from that of the vacuole. Tissue NH4+ concentrations of rice leaf using a continuous-flow auto analyzer were 11.12mmol/L. NH4+ selective microelectrode offered technical support for the study of the assimilation and utilization of NH4+ in rice.