Background <p>The search for alternative aqua feed ingredients is continuous and the costliest ingredients are replaced to cut down the feed cost and make the aquaculture venture a profitable one. A 60-day feeding trial was conducted to determine the effects of substituting groundnut oil cake (GNOC) with <i>Prosopis cineraria</i> seed meal (PSM) on the growth, physio-metabolic, and immunological responses of <i>Labeo rohita</i> fingerlings. Four iso-nitrogenous (30.36% crude protein) and iso-caloric (17.64&#xa0;MJ GE kg-1) diets were formulated with varying inclusion levels: 0, 50, 75, and 100&#xa0;g PSM kg⁻¹, denoted as Cs, PSM₅, PSM_7.5, and PSM₁₀, respectively. One hundred twenty (120) fingerlings (average weight 12.70 ± 0.02&#xa0;g) were randomly distributed in four groups in triplicates using a completely randomized design.</p> Results <p>Increasing dietary PSM levels enhanced growth and nutrient utilization indices, with the most significant improvement observed in the PSM_7.5 and PSM₁₀ groups. Intestinal protease enzyme activities improved significantly (<i>P</i> &lt; 0.05) with higher dietary PSM levels, while lipase and amylase activities remained unaffected. Similarly, the activity of the protein (aspartate aminotransferase and alanine aminotransferase) and carbohydrate metabolic enzymes (lactate dehydrogenase and malate dehydrogenase) significantly (<i>P</i> &lt; 0.05) elevated in higher PSM-fed groups. However, carcass composition, oxidative stress enzymes (superoxide dismutase and catalase) activity, and haematological biochemical parameters remained unaffected (<i>P</i> &gt; 0.05) due to feeding of varying levels of PSM.</p> Conclusion <p>PSM can be incorporated up to 100&#xa0;g&#xa0;kg⁻¹ as a substitute for GNOC in <i>L. rohita</i> diet without compromising growth and immunity. The outcome of this study may reduce the cost of aquafeed for carp culture in the context of sustainable aquaculture practices.</p>