It is difficult that burnup determination of natural uranium fuel as to its lower burnup and less fission products. 137Cs was selected as the fuel burnup monitoring nuclide. High performance liquid chromatography (HPLC) was used to separate uranium in the solution of fuel elements. Isotope dilution thermal ionization mass spectrometry (ID-TIMS) was used to determine the concentration of uranium in the fuel. Fission product-137Cs activity concentration was measured by high sensitivity gamma ray spectroscopy. High performance liquid chromatography separation, mass spectrometry determination, sample mixing calculation, 137Cs point source preparation, data correction process were introduced. Burnup of 3 natural fuel rods was determined in this paper.It is difficult that burnup determination of natural uranium fuel as to its lower burnup and less fission products. 137Cs was selected as the fuel burnup monitoring nuclide. High performance liquid chromatography (HPLC) was used to separate uranium in the solution of fuel elements. Isotope dilution thermal ionization mass spectrometry (ID-TIMS) was used to determine the concentration of uranium in the fuel. Fission product-137Cs activity concentration was measured by high sensitivity gamma ray spectroscopy. High performance liquid chromatography separation, mass spectrometry determination, sample mixing calculation, 137Cs point source preparation, data correction process were introduced. Burnup of 3 natural fuel rods was determined in this paper.