SummaryNanoliposomes (NL) containing different levels (1%, 2%, and 5%; w/w) of EPA/DHA‐enriched oil (n3FAO) from skipjack tuna eyeballs were prepared using 1%, 2.5%, and 5% (w/v) of soy phosphatidylcholine lecithin (SL) as a wall material. Then, the impact of ultrasonication at different amplitudes (40% and 50%) and sonication time (5 and 10 min) on encapsulation efficiency (EE) of selected NL was also investigated. NLs prepared without ultrasonication treatment using 2.5% SL and 2% n3FAO (WU‐NL) showed the highest EE (88%) than the remaining concentrations. When ultrasound at 40% amplitude for 5 min was applied, NLs (US‐NL) having the maximum EE of 98% were achieved. Ultrasonication employed at all conditions showed increased particle size (31.6–67.2 nm) than the WU‐NL (22.8 nm). Microstructure suggested the formation of particles with smooth surfaces and homogenous distribution. When NL powders were incorporated into the fresh cow milk at various levels (2.5% and 5.0%; w/v), likeness for aroma, mouthfeel, taste, and aftertaste slightly decreased, especially at higher levels (5%) for all samples. Nevertheless, the likeness score remained within acceptable levels (≥5.0). The fatty acid profile of the milk fortified with 2.5% (w/w) NLs showed a significant increase in polyunsaturated fatty acids (PUFAs), including EPA and DHA. Therefore, the findings indicated that ultrasonication improved the EE of liposomes, which could be successfully incorporated into milk and other food items with a lower content of PUFAs, boosting their nutritional profile while maintaining their taste and sensory attributes.