Ribonucleic acid (RNA) and its degradation products are widely used in the food industry. In this study, we constructedSaccharomyces cerevisiaemutants withFHL1,IFH1,SSF1, andSSF2overexpression andHRP1deletion, individually to evaluate the effect on RNA production. The RNA content of recombinant strains W303-1a-FHL1, W303-1a-SSF2, and W303-1a-Delta HRP1 was increased by 14.94%, 24.4%, and 19.36%, respectively, compared with the RNA content of the parent strain. However, W303-1a-IFH1 and W303-1a-SSF1 showed no significant change in RNA production compared with the parent strain.IFH1andFHL1encode Ifh1p and Fhl1p, respectively, which combine to form a complex that plays a key role in the transcription of the ribosomal protein (RP) gene. Ssf2p, encoded bySSF2, plays an important role in ribosome biosynthesis and Hrp1p is a negative regulator of cell growth inS. cerevisiae. Subsequently, a high RNA production strain, W112, was constructed by simultaneously overexpressingFHL1,IFH1, andSSF2and deletingHRP1. The RNA content of W112 was 38.8% higher than the parent strain. The growth performance,RPtranscription levels, and rRNA content were also investigated in the recombinant strains. This study provides a new strategy for the construction ofS. cerevisiaestrains containing large amounts of RNA, and it will make a significant contribution to progress in the nucleic acid industry.