The determination of dissolved oxygen (DO) in natural waters is critically important for assessing water quality, understanding the diffusion of atmospheric gases, the circulation of water masses, and studying biological and chemical reactions. The Winkler method is a well-established and conventional technique for measuring DO. However, the titration method and spectrophotometric method improved based on the Winkler principle require a significantly large amount of manual operations, multiple procedural steps, dispersed hardware, in addition to the necessity of conducting calculations for accurate results. To address these challenges, a process automation and software integration system has been developed to automate the manual operations for measuring DO. A 466 nm wavelength LED light source was used to construct a custom single-wavelength spectrophotometric detection system. Reagents and standard solutions were introduced using multiple syringe pumps, while the temperature of the flow cell was regulated with a cooling chip. Software was implemented to automate the process and facilitate data processing. Parallel samples were prepared only after pure water had been bubbled for at least 10 hours. With a total of 41 parallel samples measured on a single day, a repeatability of 0.15% relative standard deviation (RSD) was achieved. Over eleven consecutive days of measurements, the reproducibility was 0.5%. Comparison of parallel samples showed that the deviation between this automated method and the conventional manual spectrophotometric method for determining DO was within 2 μmol/L. The relative error of the determined value was 0.65%, as calculated by comparing the measured values with the theoretical oxygen concentration of saturated water at 24.4°C. The limit of detection of the developed device was determined to be 6 μmol/L by means of the standard curve calculation method.