AbstractThe current study aimed to optimize the process for ready‐to‐eat extruded fish powder preparation and to ascertain the impact of two methods on fatty acid profiles. For the investigation, Labeo rohita (Rohu) fish was first minced, extruded, and microwave vacuum‐dried. The results show that the yield for extruded fish powder (EFP) fluctuated from 22.32% to 29.07%. The maximum retention for docosahexaenoic acid (DHA), arachidonic acid (AA), and eicosapentaenoic acid (EPA) was 3.24 ± 0.08 g/100 g lipids, 2.74 ± 0.05 g/100 g lipids, and 1.24 ± 0.09 g/100 g lipids, by using different extrusion parameters. Moreover, nonsignificant changes were observed during 0 days, and 1 and 3 months of storage (at 4°C and 25°C) for DHA, AA, and EPA, whereas significant results were recorded for the samples stored for 6 months at 25°C. Also, the maximum peroxide value (PV) and thiobarbituric acid reactive substance values (TBARS) were 1.72 ± 0.04 meq/kg fat and 0.135 ± 0.008 mg malondialdehyde/kg fat. It is anticipated from the outcomes that the study will be helpful to prepare value‐added food products in future studies.