Standardized parts can be efficiently assembled into novel biological systems using the three antibiotic (3A) system, ensuring the reusability of components and repeatability of experiments. In this study, we created the 3A expression system for easy construction of gene expression cassettes inCorynebacterium glutamicum(C. glutamicum), which was applied to screen combinations of promoters and signal peptides for improved secretedrhv3production. We first obtained three strong promoters P-2252, P-odhi, and P(yweA)from all of promoters, which drive the highest expression of green fluorescent protein (egfp). The three promoters were then assembled with different signal peptides to generate a series of constructs using the 3A expression system developed in this study, from which the highest activity ofrhv3reached 3187.5 ATU/L of P-yweA-CspA-rhv3. Further increased production of rhv3achieved large-scale fermentation using 5-L jar bioreactor, with the highestrhv3accumulation 1.21 g/L obtained after 40 h of cultivation, which is higher than 0.95 g/L reported inE. coli. To the best of our knowledge, this is the first report ofrhv3secretory expression inC. glutamicum, which could be applied for the production of other recombinant proteins with significant applications. Key points center dot We have exploited a 3A system for the genetic manipulation in C. glutamicum. center dot We constructed element libraries for assembling standard sequence in C. glutamicum. center dot The secreted expression of rhv3 was realized by 3A system in C. glutamicum.