Docosahexaenoic acid (DHA) is a potential regulatory substance for flesh quality of fish, while the related evaluation is still barely. In this study, the effects of DHA-enriched diets on the flesh quality of freshwater fish (Megalobrama amblycephala) were investigated systematically. The sub-adult M. amblycephala were randomly fed with control diet (CON), 0.2% DHA diet (DL) or 0.8% DHA diet (DH). After 12-week feeding trial, the DH group flesh had higher concentrations of essential amino acids and polyunsaturated fatty acids compared to the CON group. Meanwhile, the hardness, springiness, shear force and moisture-holding capacity, as well as the values of umami, richness and sweetness were also improved by DH. The non-targeted metabolomics analysis revealed the key metabolites that may have significantly positive influence on flavor. Collectively, the diet supplementation with 0.8% DHA could achieve the improvement of the flesh quality in terms of nutritional value, texture and flavor in freshwater fish.