To extract gold from a low-grade(13.43 g/t) and high-sulfur(39.94wt% sulfide sulfur) Carlin-type gold concentrate from the Nibao deposit, Guizhou, a bio-pretreatment followed by carbon-in-pulp(CIP) cyanide leaching process was used. Various methods were used to detect the low-grade gold in the concentrate; however, only time-of-flight secondary-ion mass spectrometry(TOF-SIMS) was successful. With bio-pretreatment, the gold recovery rate increased by approximately 70.16% compared with that obtained by direct cyanide leaching of the concentrate. Various attempts were made to increase the final gold recovery rate. However, approximately 20wt% of the gold was non-extractable. To determine the nature of this non-extractable gold, mineralogy liberation analysis(MLA), formation of secondary product during the bio-pretreatment, and the preg-robbing capacity of the carbonaceous matter in the ore were investigated. The results indicated that at least four factors affected the gold recovery rate: gold occurrence, tight junctions of gold-bearing pyrite with gangue minerals, jarosite coating of the ore, and the carbonaceous matter content.