Abstract: Readily oxidizable organic carbon is the most active fraction of soil organic carbon, which deeply participates in the dissolution, migration, transformation, absorption and other processes of nutrient elements in the soil. Accurately determining the content of readily oxidizable organic carbon in soil is of great significance for exploring the circulation of carbon elements and the migration and transformation of nutrient elements in the soil. Since the concept of readily oxidizable organic carbon was proposed, the determination method involves using 25 mL of 333 mmol/L KMnO₄ to oxidize soil samples containing 15 mg of carbon. The measurement is made by observing the change in KMnO₄ concentration at 565 nm. Due to the non-fixed solid-liquid ratio during oxidation and the unclear oxidation time, there are significant differences among different researchers adopting this method. As a result, the consistency and validity of the determination results are difficult to guarantee. In this study, the distribution characteristics of readily oxidizable organic carbon in soil aggregates were investigated through particle size fractionation experiments, and the optimal particle size for sample preparation was identified to improve the oxidation efficiency of KMnO₄. Through experiments on solid-to-liquid ratio and oxidation time, the sample processing procedure was refined, thereby enhancing the stability of the determination results for readily oxidizable organic carbon. A method for determining readily oxidizable organic carbon in soil was established by spectrophotometry with constant-temperature shaking. Surface soil samples with different land use types were collected from Yunnan laterite, northeast black soil, Shaanxi loess, and Sichuan purple soil regions to conduct experimental condition optimization and method applicability studies. After optimization, the method detection limit (MDL) was 0.021%, and the method precision (RSD, n = 6) was ≤8.56%. This method is applicable for the determination of soil readily oxidizable organic carbon in projects such as natural resource surveys and ecological restoration of territorial space.