Abstract: The medical isotope test reactor that utilizes uranyl nitrate solution as its fuel source has certain advantages when it comes to the production of ⁹⁹Mo. This method has emerged as an important developmental direction for the generation of medical isotopes. Low-enriched uranium fuel is the trend of research reactor development, and the ⁹⁹Mo separation process of the medical isotope test reactor under low-enriched uranium conditions is crucial. In this study, a comprehensive analysis was conducted on the ⁹⁹Mo separation and purification process using a medical isotope test reactor that utilizes low-enriched uranium fuel. The research primarily focused on the development and optimization of a novel method involving a combination of spherical alumina columns, an α-benzoin oxime column, and an activated carbon column. The outcomes of this research are promising, as the combination of these three columns facilitated an effective extraction, separation, and purification of ⁹⁹Mo. Moreover, through the production verification of the separation of Mo in the low-enriched uranium simulated fuel solution by using this process, the recovery rate of Mo is 75.7%, and the impurities also meet the requirements. This process improves the production rate of ⁹⁹Mo of the medical isotope test reactor under low-enriched uranium fuel conditions, lays a foundation for the low-enrichment of the medical isotope test reactor, and has great application value.