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Seeking alternatives to the depleting fish oil are crucial for marine fish aquaculture, which is currently dependent on fish oil as the primary source of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs). Five isonitrogenous (46% crude protein) and isolipidic (16% crude lipid) feed diets (FO, ISO2.9, ISO4.8, ISO6.7, ISO8.6) were formulated by partially replacing fish oil with microalgae Isochrysis galbana. These diets were fed to triplicate tanks of Trachinotus ovatus (mean initial weight 1.92 g) for 80 days. This work demonstrates that a moderate inclusion (around 4.5-5.0 wt%, equivalent to the replacement of 24-26 wt% fish oil) of I. galbana biomass in fish diet improves fish growth performance, lipid deposition and enhances total n-3 fatty acids, DHA, and EPA contents in neutral and polar lipids (PLs) of fish muscle and liver of T. ovatus. The results disclosed in this study suggest that I. galbana microalgae represents a potential high-quality substitute for fish-based ingredients in aquaculture feeds, which can be a promising sustainable solution to resolve the depleting fish oil resource in a cost-effective manner. [ABSTRACT FROM AUTHOR]