Abstract: Ion-Selective Microelectrode (ISME) technology has widely been used to evaluate the plant electrical activity in mesoscopic space-time scale, including dynamic measurements on extracellular ion fluxes and intracellular ion activities. But the interference of electromigration or membrane potential changes has posed a great challenge on the traditional single-barreled ISME during the electrophysiological experiments, particularly on the accuracy of measured data. In this study, a double-barreled ISME monitoring scheme was proposed to eliminate the influence of potential drift on the measurement for more accurate information of ion flux. A simple and rapid preparation process of double-barreled ISMEs fabrication was also developed to widen the application of ion-selective electrode technology. Septum Theta (a borosilicate glass capillary with two cavities) was used to fabricate the improved double-barreled microelectrode during preparation, particularly on the silanization and liquid ion exchanger filling in the microelectrode tip of double-barreled ISME. The experimental measurements were conducted for the Nernst slope and response time of double-barreled ISMEs. A standardized feasibility plan was provided for the preparation and performance testing of double-barreled ISMEs. Experimental results showed that the optimal silanization temperature, dimethyldichlorosilane dosage, and silanization time were 150 ℃, 45μL, and 90 min, respectively, when the double-barreled microelectrodes were silanized by the steam silane. In this case, the double-barreled ISME that filled 100 μm liquid ion exchanger in the tip was reciprocated in the test solution at a frequency of 0.5Hz for 30min, where the remaining amount of liquid ion exchanger was observed under the microscope. The remaining amount of liquid ion exchanger was (100±0) μm in the double-barreled potassium ion, calcium ion, and hydrogen ISMEs' tip. In double-barreled chlorine ISMEs, the remaining amount of liquid ion exchanger in the tip was (90±8.2) μm. The Nernst slopes of double-barreled hydrogen ion, potassium ion, calcium ion, and chlorine ISMEs were 54.08, 56.51, 27.08, and -58.80 mV/dec, respectively. The measured Nernst slope of ISMEs was more than 90% of the theoretical value suitable for the requirements of the application. The response time of ion-selective electrodes with different liquid ion exchangers was between 0.2 and 0.42 s. Therefore, 0.5 Hz was a reasonable vibration frequency for the measurement of extracellular ion fluxes with the double-barreled ISMEs. The experimental results demonstrated that the double-barreled ISME developed by the improved preparation can well meet the requirements of ISME technology, thereby effectively capturing the extracellular ion fluxes or intracellular ion activities of plant cells. Consequently, the facile preparation of double-barreled ISME can be expected to greatly reduce the experimental difficulty of ISME fabrication. The finding can also provide a great contribution to acquire experimental data of plant physiological detection and applications in agricultural engineering, crop breeding, physiological stress tolerance, and cellular nutrition.