The rapid development of Eucalyptus grandis plantations has generated substantial economic benefits. Still, long-term monoculture with intensive management has also triggered a series of ecological problems, such as soil degradation and biodiversity loss. Conversion pure Eucalyptus grandis forests into mixed forests represents a crucial approach for enhancing soil fertility, restoring soil quality and achieving sustainable plantation management, which is particularly important for forest ecological conservation and economic development. However, the impacts of constructing mixed forests after clear-cutting of Eucalyptus grandis on soil phosphorus fractions and availability remain poorly understood. Here, we conducted a field experiment to examine the effects of constructing mixed forests on soil phosphorus fractions and availability in different soil layers. Four forests were selected in the replacement and transformation demonstration site of E. grandis pure forests in Shawan District, Leshan City, Sichuan Province, i.e., Cunninghamia lanceolata+Phoebe zhennan+Prunus persica 'Atropura' mixed forest, P. persica 'Atropura' + Malus × Micromalus mixed forest, Cinnamomum camphora+Metasequoia glyptostroboides mixed forest, and E. grandis pure forest. Results indicated that: (1) The construction of mixed forests increased soil microbial biomass in both 0-20 cm and 20-40 cm soil layers, with the most significant effect observed in the C. camphora+M. glyptostroboides mixed forest. (2) The construction of mixed forests significantly increased the activity of leucine aminopeptidase in the 0-20 cm soil layer, while significantly decreased the activities of β-glucosidase in the 0-20 cm soil layer and N-acetyl-glucosidase in the 20-40 cm soil layer. (3) The construction of mixed forests significantly increased the contents of soil active phosphorus and stable phosphorus in both 0-20 cm and 20-40 cm soil layers, and with the most significant effect observed in the C. camphora+M. glyptostroboides mixed forest in the 0-20 cm layer. (4) The regulatory pathways of establishing mixed forests on soil phosphorus fractions varied across soil layers: soil microbial biomass nitrogen and pH dominated in the 0-20 cm layer, whereas soil total nitrogen and soil organic carbon dominated in the 20-40 cm layer. In conclusion, the construction of mixed forests (especially C. camphora+M. glyptostroboides mixed forest) could improve the content and availability of soil phosphorus fractions and availability across different soil layers through modifying soil microbial biomass, soil enzyme activities, and soil physicochemical properties in the soil profiles. The results of our study can provide a scientific basis for the transformation of pure plantation such as E. grandis pure forests.