Abstract: Al₂O₃ chromatography is usally used for separation and purification of ⁹⁹Mo in the production process of fission ⁹⁹Mo. To establish the separation process for fission ⁹⁹Mo from low enriched uranium (LEU) target by Al₂O₃ chromatography, the effect of ⁹⁹Mo adsorption on acidic Al₂O₃ with different conditions of time, temperature, concentration of nitric acid and pretreatment method of Al₂O₃ were studied. Decontamination effectiveness of major impurities including Sr, Ru, Zr, Te, Cs and I with Al₂O₃ chromatographic method was evaluated. From experimental results, strong adsorption (more than 99%) of Mo on Al₂O₃ in 0.05-0.1 mol/L HNO₃ solution was observed and no adsorption happened in NH₄OH solution. The Al₂O₃-C activated at 500 ℃ for 3 h had a larger specific surface area than those of Al₂O₃-B activated at 150 ℃ for 3 h and Al₂O₃-A pretreated at 60 ℃. In addition, Al₂O₃-C had a better adsorption performance for Mo than Al₂O₃-B and Al₂O₃-A in HNO₃ solution with concentration greater than 0.1 mol/L, the recovery yield of Mo was more than 90% using Al₂O₃ chromatography. The decontamination effectiveness of the impurities Ru, Sr, Zr, Te and Cs was more than 99.99% and more than 92% of ¹³¹I could be removed by Al₂O₃ chromatography. Al₂O₃ has high adsorption rate, fast adsorption rate and large adsorption capacity for Mo in HNO₃ solution, which can effectively remove impurities from fission ⁹⁹Mo. It is suitable for the separation process of fission ⁹⁹Mo from low enriched uranium target.