Background <p><i>Saccharomyces cerevisiae</i> has been extensively employed as a host for the production of various biochemicals and recombinant proteins. The expression systems employed in <i>S. cerevisiae</i> typically rely on constitutive or galactose-regulated promoters, and the limited repertoire of gene expression regulations imposes constraints on the productivity of microbial cell factories based on budding yeast.</p> Results <p>In this study, we designed and characterized a series of allantoin-inducible expression systems based on the endogenous allantoin catabolic system (DAL-related genes) in <i>S. cerevisiae</i>. We first characterized the expression profile of a set of DAL promoters induced by allantoin, and further combined with the galactose-inducible (GAL) system to create a highly responsive genetic switch that efficiently amplifies the output signals. The resulting allantoin–GAL system could give a ON/OFF ratio of 68.6, with 6.8-fold higher signal output over that of direct P_DAL2-controlled gene expression. Additionally, when a centromeric plasmid was used for EGFP expression, the ON/OFF ratio was increased to &gt; 67.2, surpassing the EGFP expression levels driven by the DAL2 promoter. Subsequently, we successfully demonstrated that allantoin–GAL system can be used to effectively regulate carotenoid production and cell flocculation in <i>S. cerevisiae</i>.</p> Conclusions <p>In summary, we characterized several allantoin-inducible DAL promoters from budding yeast and further developed a layered allantoin–GAL system that utilizes the DAL2 promoter to regulate the galactose regulon in budding yeast. The resulting allantoin–GAL system could give an impressive ON/OFF ratio that surpassed the traditional P_DAL2-controlled gene expression. It is anticipated that utilizing our allantoin-inducible system in budding yeast with allantoin as the alternative nitrogen source might favor the low-cost production of biochemicals and pharmaceuticals.</p>