Abstract: The oxygen isotopic ratio will be recognized as a possible tool for identification of the origin of seized uranium oxides in nuclear forensics. The curve of mass calibration was determined according to the measuring parameter changes. Other parameters were optimized and determined by several studies, such as the intensity of primary ion, measuring time and mass resolution. The method was established to analyze the oxygen isotopic ratio in uranium oxide by SIMS. The isotopic ratio of oxygen in practical samples was measured by SIMS and the relative standard deviation (RSD) is less than 1.6%. The RSD is satisfied to trace the source of uranium oxide in nuclear forensics.