The chemical process involved in the separation and purification of 99Mo from the liquid fuel of a water-boiler reactor (WBR) has been established. The process includes separation of 99Mo from uranium sulfate fuel solution with α-benzoin oxime precipitation and purification of 99Mo by chelating ion exchanger, alumina, and calcium phosphate hydroxide as adsorbents. Recycling of uranium fuel solution to WBR was performed by purification with anion exchanger and hydrogen peroxide precipitation. Parameters affecting the recovery yield and purity of 99Mo and uranium in each process step were examined by batch study. Synthetic fuel solution experiments under the established optimum process conditions were also carried out. The experimental results conclude that the overall recovery of 99Mo product solution is 81.0±1.5% and that its radionuclide purity is quite satisfactory for the preparation of 99mTc generator for nuclear medical diagnosis applications. The recovery yield of uranium fuel was found to be 98.5±0.3%.