Accurate measurement of the oxygen isotopic composition of dissolved phosphate (δ18OP) 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 δ18OP 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 δ18OP is better than 0.3‰, demonstrating high reliability and accuracy. This new method was used to analyze the δ18OP of different P fractions in sediments of a eutrophic lake in southwestern China. The preliminary results indicated that the δ18OP 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. Display omitted •A five-step method for the analysis of δ18OP of different P fractions in freshwater sediments was established.•Use of non-phosphate activated carbon powder can remove organic matter in NaHCO3-P and NaOH-P extracts more easily.•Coprecipitation of Fe(OH)3-PO43- can remove most of metals and Cl- in HCl extract efficiently at the beginning.•We provide a powerful tool for investigating the sources and biogeochemical cycle of P in freshwater sediments.