New materials with a low environmental load are expected to be generated through synthetic biology. To widely utilize this technology, it is important to create cells with designed biological functions and to control the expression of multiple enzymes. In this study, we constructed a cell-free evaluation system for multiple protein expression, in which synthesis is controlled by T7 promoter variants. The expression of a single protein using the T7 promoter variants showed the expected variety in expression levels, as previously reported. We then examined the expression levels of multiple proteins that are simultaneously produced in a single well to determine whether they can be predicted from the promoter activity values, which were defined from the isolated protein expression levels. When the sum of mRNA species is small, the experimental protein expression levels can be predicted from the promoter activities (graphical abstract (a)) due to low competition for ribosomes. In other words, by using combinations of T7 promoter variants, we successfully developed a cell-free multiple protein synthesis system with tunable expression. In the presence of large amounts of mRNA, competition for ribosomes becomes an issue (graphical abstract (b)). Accordingly, the translation level of each protein cannot be directly predicted from the promoter activities and is biased by the strength of the ribosome binding site (RBS); a weaker RBS is more affected by competition. Our study provides information regarding the regulated expression of multiple enzymes in synthetic biology.