Abstract: This paper selects representative series of soil samples (with gradient changes in different physicochemical indicators) from soil standard reference materials and related candidate materials for effective iron component analysis, to study the matrix factors of soil properties affecting the changes in iron availability. By examining the relationship between changes in soil pH, organic matter, cation exchange capacity, calcium carbonate content, and iron availability, the matrix factors causing changes in iron availability (soil physicochemical properties and iron mineral structure) are determined. Using path analysis, the correlation coefficients between soil pH, organic matter, cation exchange capacity, and iron availability are analyzed. The results show that soil pH is negatively correlated with iron availability, with a correlation coefficient of -0.1885; organic matter and cation exchange capacity are positively correlated with iron availability, with correlation coefficients of 0.236 and 0.1959, respectively. The influence on iron availability is in the order: organic matter > cation exchange capacity > pH. In addition, through different existing states of iron in the tested samples, it is further discussed that the mineral structure of iron is a key factor determining its behavior. This study can not only provide direct solutions for addressing iron deficiency or iron toxicity in agricultural production, but also deepen the understanding of element cycling in the soil-plant system; providing a scientific basis for sustainable agriculture, environmental protection, and responding to global changes.