Abstract: The development, preparation, working principle and application of ion selective microelectrode were introduced. This technology, developed with the development of electrophysiology, was used originally for chemistry and electrochemistry analyses. Its preparation and working principle included the following aspects: 1) The preparation of a double-barred microelectrode included several steps such as making sensor, pulling electrode, backfilling, silanization, calibration and so on. 2) Working principle of a double-barred microelectrode was due to the movement of charged ions resulting from concentration differences between electrode and inside the cell. The sensor had selective permeability to certain ion and produced membrane potential, with which existed log-marker correlation and the activity of ion could be gained by modified Nernst equation. Ion selective microelectrode were widely used to illustration the physiology processes and driving energy of ions transport as well as their compartmentation, such as NH4+, NO3-, K+, Ca2+, etc. Also it could detect changes of physiological in the apoplast under different conditions and the transduction of electric signal in stress. Specificity and selectivity of ion selective microelectrode should be improved and simplified in equipment in the future so as to offer technical support in crop nutrition diagnosis and serve agriculture better.