Catfish oil (CFO) is rich in unsaturated fatty acids (UFA) which are potentially produced into margarine through the blending method with palm stearin (PS) addition. The high UFA content of CFO causes margarine to be susceptible to oxidative damage, which can also trigger the formation of trans fatty acids (TFA). An antioxidant compound in the form of avocado waste oil (AWO) that contains tocopherol is added during margarine production to inhibit oxidative damage and TFA formation in the final product. Thus, the temperature and duration of CFO extraction, avocado peel and seed ratio of AWO extraction, and CFO:PS:AWO ratio of margarine production were analyzed for their effect on CFO, AWO, and margarine characteristics. This study was conducted by several stages; CFO extraction with temperature (50, 70 and 90oC) and duration (1 and 2 hrs) treatment, AWO extraction with avocado peel and seed ratio treatment (100:0, 75:25, 50:50, 25:75, 0:100), and margarine production with CFO:PS:AWO ratio treatment (41:40:0, 36:40:5, 31:40:10, 26:40:15, 21:40:20). The results showed that the increasing extraction temperature and duration caused the decrease of CFO hydrolytic-oxidative stability. The best CFO extraction treatment was 50oC and 1 hr of extraction. The increasing avocado peel ratio in AWO extraction led to the increase of AWO antioxidant potential. The best AWO extraction treatment was the ratio of peel:seed = 75:25. The addition of AWO in margarine production for tocopherol enrichment was proven to suppress oxidative damage and TFA formation. The best margarine production treatment was the 3rd formulation (10% of AWO) with UFA-rich and trans-fat-free margarine characteristics. It was selected as the most preferred color, taste and flavor of margarine. These studies’ results are beneficial for the product development of UFA-rich and trans- fat-free margarine with good oxidative stability prepared using freshwater fish oil and plant byproduct oil.