The radioactive cesium, released from the normal operation or the accidental operation of nuclear facilities, should be regularly monitored for environmental regulatory compliance. The 135Cs/137Cs isotopic ratios, potentially useful for long-term tracking Cs transport in seawater, can be used as a tool of understanding how radionuclides are transported from different nuclear production source terms and distributed in the ocean. The ultra-high sensitive mass spectrometers (TIMS, SF-ICP-MS and TQ-ICP-MS) have been used to measure the 135Cs/137Cs isotopic ratios. However, the radiochemical separation of Cs from the seawater matrix is essential for the analysis of Cs using the mass spectrometers. An automated radiochemical procedure for the separation of Cs in seawater was developed for the analysis of 135Cs/137Cs isotopic ratios using a sequential column chromatography with AMPPAN and AG50Wx8 cation exchange resins. National Instrument’s LabVIEW is a graphical programming language and a powerful tool for the instrument control. A virtual instrument system for the automated separation of cesium isotopes was developed by the state machine of the fundamental design patterns in LabVIEW. In this study, the conceptual designs of an automated separation system of cesium isotopes, its virtual instrument system based on the LabVIEW state machine architectures and an automated radiochemical procedure were described for the purification of cesium isotopes at trace levels found in seawater discharged from the various nuclear facilities.