Ammonium thiosulfate is an alternative lixiviant for the hydrometallurgical treatment of sulfide gold ores. The present study is primarily focused on ammonium thiosulfate leaching of gold (Au) and silver (Ag) from the sulfide ore (Sunshin mine in Korea). The main chemical composition of the concentrate was Au (84 ppm), Ag (852 ppm), Fe (18.9%), Si (23.2%), and S (21.1%). The effects of various parameters on the process, such as leaching time (1–4 h), ammonium thiosulfate concentration (0.05–0.5 M), copper sulfate (CuSO4), concentration (0.05–0.25 M), solid to liquid ratio (0.2–0.5), and reaction temperature (40–60 °C) were systematically examined. Optimum Au leaching efficiency (>99%) was obtained under the following leaching conditions: 0.5 M ammonium thiosulfate with 0.05 M CuSO4 concentration, 0.2 S/L ratio at 60 °C for 2 h. The results indicate that the behavior of Ag was similar to that of Au. Almost complete dissolution of Ag occurred under following leaching conditions: 0.5 M ammonium thiosulfate with 0.05 M CuSO4 concentration at 60 °C for 4 h. This study would be useful in understanding the eco-friendly leaching systems of Au and Ag during the hydrometallurgical process of sulfide gold ore.