Copper is an essential trace element for living organisms. Copper enriched by yeast of Saccharomyces cerevisiae is regarded as the biologically available organic copper supplement with great potentiality for application. However, the lower uptake ratio of copper ions makes the production of copper enriched by yeast uneconomically and environmentally unfriendly. In this study, S. cerevisiae Cu-5 with higher copper tolerance and intracellular copper accumulation was obtained by screening of our yeast strains collection. To increase the uptake ratio of copper ions, the medium composition and cultivation conditions for strain Cu-5 were optimized systematically. A medium comprised of glucose, yeast extract, (NH4)2SO4, and inorganic salts was determined, then a novel cultivation strategy including pH control at 5.5 and increasing amounts of yeast extract for a higher concentration of copper ion in the medium was developed. The uptake ratios of copper ions were more than 90% after combining 50 to 100mg/L copper ions with 3.5 to 5.0g/L yeast extract, which is the highest until now and is conducive to the cost-effective and environmentally friendly production of bioactive copper in yeast-enriched form.