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Microalgae offer a nutritionally robust alternative to fishmeal and fish oil, helping reduce pressure on wild stocks and supporting more sustainable aquafeed production. This study explored the potential of replacing fish oil with Tetraselmis ( Tetraselmis chui ) microalgae biomass in the diet of juvenile rainbow trout (89.0 ± 1.10 g) ( Oncorhynchus mykiss ), assessing its effects on the fish’s health. A control diet containing 53% crude protein and fish oil (FO) was modified by replacing FO with Tetraselmis at three graded inclusion levels: 33% (Tetra33), 66% (Tetra66), and 100% (Tetra100). The 84-day feeding trial evaluated key growth parameters, biochemistry, liver and intestinal histo-architectures, and immune-antioxidant gene expression profiles of the experimental fish. Time-series analyses of growth performance revealed no significant treatment effects from day 14 to day 70, except at the 84-day biomass sampling. The FO (7321.65 ± 60.03g) attained a significantly greater final weight (FW) than Tetra33 (6984.70 ± 86.15g) and Tetra100 (6823.93 ± 160.42g), while remaining statistically similar to Tetra66 (7051.77 ± 107.30g). Likewise, weight gain (WG) of the FO group (5519.65 ± 57.16g) exceeded that of the Tetra100 group (5043.93 ± 142.09g) but did not differ significantly from the Tetra33 (5220.70 ± 73.95g) and Tetra66 (5281.77 ± 110.86g). The feed conversion ratios (FCRs) and specific growth rates (SGRs) of the Tetra groups were comparable to the FO control. Dietary variation did not elicit significant changes in leukocyte distribution, biochemical indices, or gene expression patterns across Tetra groups relative to the FO. Similarly, the histological analysis revealed that Tetraselmis dietary inclusions did not trigger inflammatory reactions in hepatic or intestinal tissues in the Tetra groups compared to the FO. Minor but inconsequential histological modifications were noted, such as moderated sinusoid dilation in the liver and slight changes in intestinal villi of Tetra33 fish. Health biomarker analyses indicated that replacing fish oil with Tetraselmis preserved physiological homeostasis, whereas 66% replacement (Tetra66) yielded the best growth performance compared to FO. However, longer feeding trials are necessary to confirm long-term health and nutritional outcomes.