Glycerol oxidation over a Pt catalyst can be effectively enhanced by a Bi additive in terms of activity and 1,3-dihydroxyacetone (DHA) selectivity. However, this catalyst system can suffer from serious deactivation. Here, a Bi-promoted Pt catalyst was developed to selectively produce DHA by maintaining the glycerol solution with Bi(NO₃)₃·5H₂O to meet the continuous operation of practical applications. The catalyst deactivation mechanism was investigated in terms of leaching, over-oxidation, agglomeration and poisoning. It was ascertained that the catalyst deactivation was due to the adsorption of chelating intermediates on the catalyst surfaces. A regeneration method by post-heat-treatment at 200–300 °C could remove the intermediates and regenerate the catalyst, allowing recycling for at least 5 runs without clear catalyst deactivation.