Saccharomyces cerevisiae, a generally recognized as safe (GRAS) microorganism, is regarded as an industrial workhorse for chemical and recombinant protein synthesis. In the biotechnological industry, multi-copy gene integration represents an effective strategy to maintain a high-level production of recombinant proteins-of interest, and to assemble multi-gene biochemical pathways. In the present study, we first created a copper-sensitive yeast strain by abolishing the CUP1 gene (encoding metallothionein that binds copper ions). Subsequently, we harnessed the CUP1 as a selection marker and combined the δ sites for multi-copy gene integration in S. cerevisiae. With the newly developed CUP1-tagging platform, multi-copy chromosomal integration of enhanced green fluorescent protein (eGFP) expression cassette was achieved, and the engineered strains remained quite stable after successive rounds of passaging. Taken together, we envision that the CUP1-tagging system would be a robust and useful method for protein overproduction and metabolic engineering applications in budding yeast.