Understanding codon usage has been vital for optimizing biotechnological applications across various domains. In response to this necessity, this review examines the impact of codon usage on protein expression systems, highlighting strategies to enhance yield and solubility in prokaryotic and eukaryotic hosts. We have discussed the role of codon usage in vaccine development, emphasizing how tailored codon sequences improve antigen expression and immune responses. In therapeutic protein production, optimizing codon preferences is shown to increase yields and reduce misfolding, facilitating the cost-effective manufacture of complex proteins. The review further explores the significance of codon usage in gene therapy, metabolic engineering, and synthetic biology, where precise codon design can drive innovations in genetic circuits and metabolic pathways. Additionally, we addressed emerging technologies and methodologies that enhance our understanding of codon optimization, paving the way for future advancements in biotechnology. By integrating insights from most recent studies, this paper underscores the importance of codon usage and its optimization, highlighting its transformative potential in enhancing the efficacy and scalability of biotechnological processes. Overall, we propose future directions for research, advocating for a multidisciplinary approach that combines computational tools and experimental validation to unlock the full potential of codon usage in biotechnology.