Tuna oil is easily subject to oxidation due to the high susceptibility to oxygen of the highly unsaturated fatty acids. To overcome this drawback, chitosan-based encapsulation may help to maintain stability and nutritional value of tuna oil. The overall objectives were to develop a new encapsulation process for tuna-oil oil and to evaluate the properties of the encapsulated powder. Encapsulation was conducted in three stages; (1) dissolution of wall material, mixing with core and emulsification; (2) atomization into a reactive broth; and (3) Freeze drying. The effects of preparation variables such as concentration and ratio of wall materials components (chitosan, maltodextrim, and whey protein isolate), tuna oil concentration (10%, 20%, 30%), amplitude of atomizer (50, 55, 60, 65) were evaluated. The emulsion was evaluated for droplet size and stability while antioxidant activity was measured over time of storage at 25 C and 50C. The powders were characterized after freeze drying in terms of tuna-oil content, EPA and DHA fatty acids composition, and microstructure of formed microspheres. The results showed that the optimum concentration of chitosan-maltodextrin and chitosan-whey protein isolate ratio were found to be 1:10 and 1:1, respectively. Emulsification process was at 5,000 rpm, 30 minutes followed with atomization at the amplitude 55. By varying the preparation conditions, dispersed phase particles in the emulsions were significantly different in size (P<0.05) with thechitosan-maltodextrin wall material giving the smallest particle size and the highest emulsion stability. There was significantly improved oxidative stability of all samples with or without -topherol compared to bulk tuna-oil. The encapsulated powder had EPA and DHA levels > 100 mg/g. Moisture content, water activity and encapsulation efficiency were acceptable. Scanning Electron Microscope showed particle with no pores or surface oil droplets. The results suggest that chitosan mixed with maltodextrin or whey protein isolate have the potential to be used as the wall material for encapsulating tuna oil or other oils using ultrasonic atomizer. However, all condition that can increase the rate of oxidation must be avoided in every step of the process because once the oil is oxidized the process can not be reversed by microencapsulation.