Supercritical carbon dioxide (SC-CO2) extracted Atlantic salmon frame bone oil (SFBO) was used for Eicosapentaenoic acid and Docosahexaenoic acid (EPA-DHA) concentrate production by urea complexation. Urea/ fatty acids (2.5 to 4.0 w/w), crystallization temperature (-24 to -8°C) and crystallization time (8 to 24 h) were studied by Box-Behnken Design (BBD) to maximize EPA-DHA content. Highest EPA-DHA content was 60.63% at urea/fatty acids 4.0 w/w, crystallization temperature -15.67 °C and crystallization time 8 h. EPA-DHA concentrate showed improvement of EPA-DHA from 6.39% in SFBO to 62.34%, increase of astaxanthin content from 21.33 μg/g in SFBO to 44.69 μg/g in EPA-DHA concentrate, no residual urea and reduction of many off-flavor compounds. The EPA-DHA yield showed an inverse relation with the urea/fatty acids, whereas its concentration increased proportionally with urea/ fatty acids. Therefore, EPA-DHA concentrate produced from SFBO by urea complexation may be an efficient technique to provide ω-3 polyunsaturated fatty acids to the consumers.