The biologically-based soil phosphorus (BBP) fractionation method assesses soil P availability by considering plant rhizosphere mediated P acquisition mechanisms: root interception, organic acid complexation, enzyme hydrolysis and acidification caused by proton excretion, and is a convenient method to reflect soil P status. This study examined the effects of straw incorporation and water management on biologically-based soil P fractions and rice P uptake in a double rice cropping system in subtropical China. Four treatments were set up, that was (i) no straw incorporation with intermittent irrigation (S0W1), (ii) rice straw incorporation with intermittent irrigation (S1W1), (iii) no straw incorporation with continuous flooding (S0W2), and (iv) straw incorporation with continuous flooding (S1W2). Four kinds of biologically-based soil P fractions were measured using the BBP fractionation method, which are the P fractions extracted by CaCl₂ solution (CaCl₂—P), citric acid solution (Citrate—P), phytase and phosphatase solution (Enzyme—P), and HCl solution (HCl—P). The results showed that straw incorporation treatments significantly increased the contents of soil CaCl₂—P, Enzyme—P, and Citrate—P, compared with the corresponding no straw incorporation treatments, while continuous flooding increased CaCl₂—P and Citrate—P, compared with intermittent irrigation in the early rice season. Straw incorporation treatments significantly increased the contents of Enzyme—P and Citrate—P, compared with the corresponding no straw incorporation treatments, and continuous flooding increased CaCl₂—P, compared with intermittent irrigation in the late rice. HCl—P was reduced in rice straw incorporation with intermittent irrigation and no straw incorporation with continuous flooding, compared with the corresponding rice straw incorporation with continuous flooding and no straw incorporation with intermittent irrigation, irrespective of the early and late rice season. The main sources of available P in paddy soil were Enzyme—P and Citrate—P because of soil available P being positive correlation with Enzyme—P and Citrate—P. Straw incorporation treatments significantly reduced rice grain P uptake, compared with the corresponding no straw incorporation treatments in the early rice season, and the rice P uptake was the lowest in straw incorporation with continuous flooding. In the late rice season, straw incorporation significantly increased both grain and straw P uptake of rice, compared with the corresponding no straw incorporation treatments, and water managements had no significantly difference in P uptake of rice. Except for HCl—P, soil biologically-based phosphorus was positively correlated with total P uptake, and was negative correlation with total P uptake in the early rice season, while was positively correlated with rice P content in the late rice season. Soil Citrate—P content was the second among the four kinds of BBP fractions (only lower than HCl—P), so Citrate—P played a major role in rice P uptake. Straw incorporation is beneficial to improve soil P availability and rice P utilization in the double rice cropping system.