Abstract: The organic amendment has been proven to be an important practice for enhancing soil phosphorus availability. However, the mechanism of enhancement availability of soil phosphorus in red paddy soils after long-term milkvetch and pig manure amendment remains unclear. In the present study utilized a 40-year long-term field experiment with milk vetch and pig manure replacing chemical fertilizers to examine the effects on soil P availability and to elucidate the chemical mechanisms responsible for P enhancement. 30% or 50% of the nitrogen input by mineral fertilizers were replaced by nitrogen derived from milkvetch or manure. The sequential chemical extraction and isothermal sorption curve fitting were used to analyze the characteristics of soil P transformation. The changes of soil dissolved organic matter molecular detected by three-dimensional excitation-emission matrix fluorescence spectroscopy and Fourier transform ion cyclotron resonance mass spectrometry, and the changes in soil available cation content measured by Mehlich 3 extraction was integrated to analyze the possible mechanisms driving soil P transformation. The results showed that:1) after long-term replacement of 30% and 50% of chemical fertilizers with milkvetch and pig manure, the available P content in the soil increased by 45.3% and 52.3%, respectively. Long-term replacement of mineral fertilizers with milkvetch and pig manure significantly reduced the proportion of soil HCl-P fraction and significantly increased the proportion of soil H₂O-P+NaHCO₃-P fractions; it also significantly increased the content of available calcium and available magnesium in the soil. 2) Correlation analysis revealed a highly significant negative correlation between soil available P content and the proportion of HCl-P fraction (P < 0.01), and a significant positive correlation with the proportion of H₂O-P+ NaHCO₃-P fractions (P < 0.05). 3) Long-term replacement of 50% chemical fertilizer with milkvetch and pig manure significantly reduced the maximum P sorption capacity of the soil by 7.34%, while the proportion of humic acid-like fluorescent components and the relative abundance of lignin and condensed aromatic structures in soil dissolved organic matter were also significantly increased. These results indicate that long-term replacement of mineral fertilizer with milkvetch and pig manure promotes the dissolution of HCl-P fraction while inhibiting the fixation of phosphate by soil through sorption process, with both effects significantly enhancing P availability. The changes of molecular structure characteristics of soil dissolved organic matter plays an important role in promoting the dissolution of HCl-P fraction and inhibiting the sorption of phosphate in the soil. Overall, this study explored the mechanisms of accumulated P mobilization induced by the long-term organic substitution. Accordingly, an integrated organic-inorganic fertilization practice with milk vetch and pig manure replacing 30%～50% of chemical fertilizer nitrogen is recommended for red soil paddy fields, which can meet rice P demand by mobilizing accumulated soil P, promote the resource utilization of organic wastes, and increase SOC stocks through the input of organic components. The findings can provide a theoretical basis for the strategies design and technological development in order to promote the mineral P fertilizer reduction and nutrients efficient utilization in red soil paddy fields.