Eicosapentaenoic acid (EPA), an essential omega-3 polyunsaturated fatty acid, can promote human health, including cardiovascular protection and anti-inflammatory effects. Traditional EPA sources, such as fish oil, face challenges including overfishing, environmental contamination, and ethical concerns. Microalgae, as the primary producers of EPA in marine ecosystems, offer a sustainable and scalable alternative. However, microalgae-based EPA production faces challenges such as high costs and low efficiency. This review explores innovative strategies to reduce costs and improve production efficiency in microalgae-based EPA production, highlighting strategies to significantly enhance EPA yields through screening of high-yielding strains, biotechnology strategies, fermentation innovations, and downstream processing. Emerging technologies like CRISPR-Cas9 enable precise genetic modifications, while artificial intelligence (AI) accelerates enzyme optimization and predictive modeling of cultivation parameters. Multi-omics integration provides insights into genotype-phenotype relationships, guiding strain design. Automation and real-time monitoring driven by internet-of-things enhance scalability and reduce operational costs. The comprehensive utilization of microalgae by-products is also emphasized, enhancing economic efficiency and sustainability. The integration of advanced biotechnological and engineering tools promises to revolutionize EPA production.