Abstract: Molybdenum isotopes of ⁹⁵Mo,⁹⁷Mo, ⁹⁸Mo and ¹⁰⁰Mo are the important fission products in nuclear fuel. In order to determine the molybdenum isotope ratios of irradiated nuclear fuel element, MoO₃^- was detected by negative thermal ionization mass spectrometry (NTIMS) using SrCl₂ as an emitter. As a result, 0.1 μg Mo sample loaded onto single rhenium filament can obtain stable ion beam for about 2 h, and relative standard deviation (RSD) of molybdenum isotope ratio determination was less than 0.5‰. 7 molybdenum isotopes were measured using 5 Faraday cups with 1 peak jumping. The m/z 143 signal was collected before and after the peak jumping, respectively, and selected as the denominator of isotope ratio, which avoided the signal fluctuation error. Background level of natural molybdenum was evaluated, which indicated that background of rhenium filaments and reagents can be ignored in the process of mass spectrometry determination. Oxygen isotope interference in MoO₃^- ions was deducted by means of solving matrix equation group. The determination results of ruthenium, molybdenum and zirconium mixed samples show that the method can reduce the isobaric interferences of zirconium and ruthenium. The natural molybdenum interference in spent fuel element was deducted by data correction, and the molybdenum isotope ratios of fission product were obtained.