Freeze drying and frozen preservation way was used to preserve a moderately thermophilic culture for bioleaching of chalcopyrite concentrate. After preservation of 15 months, the cell viability rate decreases to 22% with a cell density of 7×107 mL-1. When the growth time was extended from 8 days to 14 days, cell density would increase in a large scale to about 3×108 mL-1. In the bioleaching experiments, unpreserved and preserved cultures were compared for dissolving chalcopyrite concentrate. Before 44 days, the unpreserved culture can reach a high copper extraction of about 17.4 g/L. While the preserved culture shows a rather low copper extraction, which is only 9.7 g/L. When the bioleaching time was extended to 80 days, copper extraction by preserved culture increases remarkably, and the concentration of copper finally achieves up to 18.3 g/L. On the other hand, copper extraction by the unpreserved culture does not show remarkable increase from 44th to the 80th day, and finally the total copper extraction is 19.8 g/L. As a result, total copper extraction in 80 days by preserved culture approaches that by unpreserved culture and freeze drying and frozen preservation even after 15 months does not bring much decrease of bioleaching ability.