Accurate measurement of the oxygen isotopic composition of dissolved phosphate (δ¹⁸O_P) of different phosphorus (P) fractions in lacustrine sediments is very difficult because of the influence of large amounts of impurities. In this study, we developed a five-step method for obtaining high purity Ag₃PO₄ sample for the analysis of δ¹⁸O_P of different P fractions in freshwater sediments. Sedimentary P was divided into NaHCO₃-P, NaOH-P and HCl-P by chemical sequential extraction. Pretreatment procedures for different sedimentary P fractions were improved in the following respects: 1) abandonment of the magnesium-induced coprecipitation method to avoid the introduction of impurity ions, such as Mg²⁺ and Cl⁻, and reduce the loss of P; 2) use of a small amount of non-phosphate activated carbon powder to efficiently remove organic matter in extracts of NaHCO₃-P and NaOH-P, and reduce the loss of P; 3) adjustment of the HCl-P extract pH to 4 in order to form Fe(OH)₃-PO₄³⁻ coprecipitate, thereby removing most of metals and Cl⁻. This method reduces the pretreatment steps, simplifies the operation and increases the recovery of phosphate (90.98%–96.69%). The high purity Ag₃PO₄ sample can be obtained and the repeatability and accuracy of measured δ¹⁸O_P is better than 0.3‰, demonstrating high reliability and accuracy. This new method was used to analyze the δ¹⁸O_P of different P fractions in sediments of a eutrophic lake in southwestern China. The preliminary results indicated that the δ¹⁸O_P in the sediments can be used to identify different P sources, and provide new insights into sedimentary P cycling. The method established in this study provides a powerful tool for investigating the sources and biogeochemical cycle of P in freshwater sediments.