Gold catalysts have been studied in-depth due to their unique activities for catalytic CO oxidation. Supports have intrinsic motivation for the high activity of gold catalysts. Thermally stable urchin-like CuO microspheres, which are potential support for gold catalysts, were prepared by facile solution-method. Then gold nanoparticles were loaded on them by deposition-precipitation method. The obtained gold catalysts were characterized by SEM, XRD, TEM, BET, ICP, and XPS. Their catalytic activity for CO oxidation was also evaluated. TEM results revealed that the gold nanoparticles with small sizes were highly distributed on the CuO surface in Au1.0/CuO-300. XPS observations demonstrated that the gold species in Au1.0/CuO-300 was of metallic state. Among the as-prepared catalysts, the Au1.0/CuO-300 catalyst displayed the best performance for CO oxidation and achieved 100% CO oxidation at 80 °C. It kept 100% conversion for 20 h at a reaction temperature of 180 °C, and showed good reusability after three reaction-cycles. The possible catalytic mechanism of Au1.0/CuO-300 catalyst for CO oxidation was also briefly proposed.