Carbonaceous sulfidic gold ores are commonly double refractory and thus require pretreatment before gold extraction. In this paper, the capacity of pre-bio-oxidation can simultaneously decompose sulfides or deactivate carbonaceous matters (CM) from a double refractory gold ore (DRGO) using pure cultures of A. ferrooxidans or L. ferrooxidans, and a mixed culture containing Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans was investigated. The results showed that direct thiourea leaching of the as-received DRGO yielded only 28.7% gold extraction, which was due to the encapsulation of sulfides on gold and the gold adsorption of CM. After bio-oxidation, thiourea leaching of the DRGO resulted in gold extraction of over 75–80%. Moreover, bio-oxidation can effectively reduce the adsorption of carbon to gold. XRD, SEM-EDS and FTIR analysis showed that many oxygen-containing groups were introduced on the surface of DRGO during bio-oxidation, while the C=C bond was cleaved and the O–C–O and C–N bonds were degraded, causing a decrease in active sites for gold adsorption. Moreover, passivation materials such as jarosite were formed on the surface of DRGO, which might reduce the affinity of CM for gold in solutions. In addition, the cleavage of the S–S band indicated that sulfides were oxidized by bacteria. This work allows us to explain the applicability of pre-bio-oxidation for degrading both sulfides and CM and increasing gold recovery from DRGO in the thiourea system.