A method of anion exchange high performance liquid chromatography was developed for the determination of five nucleotides，including cytidine monophosphate(CMP)，adenosine monophosphate(AMP)，uridine monophosphate(UMP)，guanosine monophosphate(GMP) and inosine monophosphate(IMP).The samples were extracted with water，then precipitated protein with precipitants.The determination of analytes was performed by HPLC on a Sax column using methanol-KH2PO4（10 mmol/L，pH 45） as mobile phase by gradient elution.The detection wavelength was set at 254 nm，and the external standard method was used for quantitation.Result showed that there were good linearities for five nucleotides in the concentration range of 0200-200 mg/L，with their correlation coefficients（r） of 0999 8-1000 0.The spiked recoveries were in the range of 955%-108% with RSDs of 10%-32%.With the advantages of good separation，wide linear range，good stability and accuracy，the method solved the matrix interference problem in sample detection based on GB 541340-2016 52，and enriched the chromatographic conditions for GB 541340-2016.