This study developed a sustainable method for gold extraction from low-grade refractory ore by combining acidic biooxidation and thiocyanate (SCN) leaching (BIOX-TC) in one system to avoid neutralization and minimize the complexity of the process. Among leachant candidates, thiocyanate was proposed as a less toxic reagent for gold leaching. Furthermore, thiocyanate requires ferric iron as an oxidant for gold extraction and ferric iron can be provided by the microorganisms during biooxidation. In the first step, acidic biooxidation was done using a mixture of acidophiles including Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Leptospirillum ferrooxidans resulting in 59.6% sulfur oxidation. A preliminary static incubation of Acidithiobacillus thiooxidans also improved biooxidation efficiency achieving higher sulfur oxidation of 87.8%. Following biooxidation, thiocyanate leaching using 0.2 M SCN was performed under three different conditions: (1) in a separate flask with 0.01 M ferric iron addition; (2) in-situ leaching, in the biooxidation flask with the mixed bacterial culture); (3) in-situ leaching in the biooxidation flask with the bacterial culture and with 0.01 M additional ferric iron. It was found that in-situ thiocyanate leaching of the biooxidized ore in the presence of the bacterial culture and without ferric iron addition resulted in the highest gold recovery (86.9%). The biooxidation mechanism and its kinetics have been extensively discussed in this paper.