Docosahexaenoic acid (DHA) (22:6n-3) is an n-3 (omega-3) long-chain polyunsaturated fatty acid (LC-PUFA) that has attracted much attention in recent years. It is indispensable in the brain and visual development of infants and young children and has many physiological functions, such as the regulation of blood lipids, inhibition of tumor growth, and prevention of Alzheimer’s disease. Fish are an important source of DHA in humans. However, with the decline of wild-capture marine fisheries and the increasing aquaculture production, the proportion of DHA obtained from farmed fish is projected to increase significantly. Recent studies suggest that genetic manipulation or selective breeding can enhance endogeous DHA biosynthesis in farmed fish by targeting key regulatory factors, potentially reducing dietary fish oil reliance while increasing DHA content in muscle. During the biosynthesis of LC-PUFAs, elongation of very long-chain fatty acids (Elovl) and fatty acid desaturases (Fads) play important roles by extending the carbon chain and introducing double bonds, respectively. This article provides an overview of the DHA biosynthesis pathways in fish and the Elovl and Fads gene family members that play key roles at various nodes, and explores the division of labor, functional compensation, and diversity of these genes. The different regulatory modes of transcription factors on the key enzyme genes mentioned above and their effects on DHA biosynthesis were analyzed at the promoter level. This article provides a theoretical reference for genetic improvement and precision breeding of the DHA content trait in farmed fish using techniques such as gene editing or molecular module design.