The 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.