<p>This review explores codon usage bias, the non-random preference for synonymous codons, as a fundamental aspect of gene regulation, translation efficiency, and genome evolution in eukaryotes. Unlike prokaryotes, eukaryotic codon usage is influenced by GC content, gene expression levels, tRNA availability, and selection pressures. Evolutionarily, mutation, genetic drift, and natural selection shape codon preferences, leading to distinct biases across unicellular and multicellular organisms. Fungi, plants, and animals exhibit lineage-specific codon usage, often linked to translational efficiency and environmental adaptation via optimized protein production. Additionally, organelle genomes such as mitochondria and chloroplasts retain unique codon patterns due to their endosymbiotic origins. Beyond evolutionary roles, codon bias impacts human health, contributing to genetic diseases, cancer progression, and neurodegenerative disorders, distinct from its applications in gene therapy and vaccine design. Advances in computational approaches, including indices like Effective number of codons (ENC), Codon adaptation index (CAI), and Relative synonymous codon usage (RSCU), along with machine learning models and specialized databases, have enhanced codon usage analysis, providing insights into gene expression and functional genomics. Despite significant progress, many aspects of eukaryotic codon usage remain unexplored, presenting new opportunities in biotechnology, synthetic biology, and genomic research. A deeper understanding of codon bias can improve translational control strategies and optimize gene expression for therapeutic and industrial applications.</p>