Discharge of acid mine drainage (AMD) from abandoned coal mines of the YuDong catchment in Kaili City, Guizhou Province, China, has severely damaged local ecological environments. In this study, a laboratory-scale dispersed alkaline substrate (DAS) was studied for the treatment of simulated AMD. The experimental conditions and reaction mechanisms were preliminarily explored. The treatment effect and variation law of vertical effluent water quality of the experimental conditions were thoroughly analysed. The results indicated that small-sized limestone (diameter 5-7 mm) having a 20:1 mixture ratio with shavings and minimum HRT of 20 hours result in increasing effluent pH from 3.5 to 6.6, achieving 66.2% and 99.1% removal of Fe and Al, respectively. There were obvious differences in each reaction layer for the removal of various pollutants from AMD along the depth by DAS, the main reaction zone was first 20-30 cm of the reaction column. The removal process of metal ions and sulfate was accompanied by bio-mineralization reaction. This test provided a valuable support for the local practical engineering applications, enriched the AMD processing technology experimental cases, and provided reference for the treatment technology of similar polluted areas.