The invention discloses a saccharomyces cerevisiae chromosome as well as a construction method and application thereof. The saccharomyces cerevisiae chromosome comprises two telomeric repeat sequences, two insulator sequences, a centromere-autonomously replicating sequence, a po130 gene and n repeated recombination boxes. Experiment results prove that auxotrophic marker genes of the saccharomyces cerevisiae chromosome are few, normal and stable growth in the situation that screening is not carried out can be realized, the expression quantity and copy number of exogenous genes in yeast are increased, the stability of exogenous genes in yeast is improved, genes integrated into a genome can be increased or reduced, and even expanded into new polygene clusters, and the rapid exogenous expression of beta carotene and violacein is successfully realized in yeast, so that a rapid feasible method is provided for yeast heterogeneous production of various exogenous approaches, and the saccharomyces cerevisiae chromosome can meet the application requirements of fields of gene engineering, metabolic engineering and synthetic biology.