In the present study, fish oil (FO) and wall material were supplemented to milk to produce spray-dried powder (SDP). Furthermore, the mandate of the study was to enlighten the effect of spray-drying (SD) operating conditions on functional and oxidative quality of produced SDP samples. Purposefully, the cow milk was supplemented with 3% FO as omega-enriched source of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) for development of milk and FO blends (MFOBs). The lecithin was used as an emulsifier and maltodextrin was supplemented as the wall material (WM) in the MFOBs. Initially, the FO, milk fat (MF), and MFOB samples were characterized for EPA, DHA, and peroxide value (PV) before the SD. The SD of MFOB samples was carried out to produce SDP samples by using a mini spray dryer. Central composite design (CCD) with face-centered rotation was used to optimize SD independent conditions such as inlet air temperature (IAT), pump speed (PS), maltodextrin percentage (MD), and needle speed (NS) in the ranges of 160–200°C, 3–9 mL/min, 10–30%, and 5–9 s, respectively. The encapsulation efficiency (EE) ranged between 89.30 and 81.57%. The EPA and DHA retentions were in the ranges of 2.19–1.87 g/100 g and 3.20–2.75 g/100 g, respectively. The highest results for responses were observed on the following conditions: IAT was 160°C, PS was 9 mL/min, MD was 30%, and NS was 9 s, respectively; the minimum values of response factors were obtained on the following conditions: IAT was 200°C, PS was 3 mL/min, MD was 10%, and NS was 5 s, respectively. The percent losses of EPA and DHA were noted in the range of 2–18%. The IAT was observed as main factor for FA reduction in SDP samples. The SDP samples were stable, and low rate of peroxide values was noted. Overall, spray drying can be potentially used to incorporate the essential fatty acids in milk to produce stable SDP for food applications.