Abstract: Activated carbon was used to absorb Xe at low temperature from atmosphere. After separation with carbon and purification with molecular sieve, purified Xe samples were attained and used for accurate isotopic measurement by gas mass spectrometry. The separation conditions of Xe using carbon and molecular sieve were studied. The R(¹³⁴Xe/¹²⁹Xe), R(¹³¹Xe/¹²⁹Xe), and R(¹³²Xe/¹²⁹Xe) from atmosphere were measured. The relative standard deviations are 0.32%, 0.15%, and 0.14%(n=3), respectively. The spent fuel shearing and dissolved tail gas was purified and measured with this method. And the burnup of the spent fuel was calculated by Xe isotope ratio. The results show that the burnup estimated by R(¹³²Xe/¹³⁴Xe) is closer to the true value than R(¹³¹Xe/¹³⁴Xe), and the deviation from the true value is about 20%.