Nowadays, due to the severe shortage of the primary metal resources used in strategic industries such as catalytic industries, their recovery from wastes such as spent catalysts of variety industries has an especial importance. The bioleaching process due to lower energy consumption, lower capital and operating costs and being more environmentally friendly than chemical leaching is a suitable method for separation of valuable metals from metal sulphide resources. The purpose of this research is to extract nickel from spent NiO-Al 2 O 3 catalyst in the presence of either a sulphide tailing or elemental sulphur. The influence of some critical parameters namely, microorganism type, ratio of spent catalyst to sulphide tailing, elemental sulphur addition, pH, solid content, type of nutrient medium, adaptation time and mechanical activation on the nickel recovery from the spent catalyst of reforming unit of Mobarakeh Steel Industry (Isfahan, Iran) was investigated. Results showed that the extraction of nickel in the presence of moderate thermophiles was more efficient than that obtained in the presence of mesophiles. Maximum nickel extraction (96%) was achieved after 31 days at the ‌‌‌spent catalyst to sulphide tailing mass ratio of 40 to 60, pH of 1, solid content of 10% (w/v) in the modified-9K nutrient medium. It was also found that the increasing the adaptation time from 40 to 120 days significantly enhanced the nickel extraction about 1.5 to 2 times. Regringing the spent catalysts by a planetary ball mill led to decrease nickel extraction around 6% likely as a result of reducing the mass transfer rate of oxygen. Analysis of the solid residue by SEM/EDX indicated the formation of jarosite precipitates on the solid surface. Results of bioleaching experiment in a 50-L stirred tank reactor showed that about 75% of nickel was extracted from the spent catalyst after 35 days at the solid content of 20% (w/v), pH of 1 and 45 ?C