Fish gelatin and its hydrolysates exhibit a variety of biological characteristics, which include antihypertensive and antioxidant properties. In this study, fish gelatins were extracted from extrusion-pretreated tilapia scales, and then subjected to analyses to determine the physicochemical properties and antioxidant activity of the extracted gelatins. Our findings indicate that TSG2 (preconditioned with 1.26% citric acid) possessed the greatest extraction yield, as well as higher antioxidant activities compared with the other extracted gelatins. Hence, TSG2 was subjected to further hydrolyzation using different proteases and ultrafiltration conditions, which yielded four gelatin hydrolysates: TSGH1, TSGH2, TSGH3, and TSGH4. The results showed that TSGH4 (Pepsin + Pancreatin and ultrafiltration < 3000 Da) had a higher yield and greater antioxidant activity in comparison with the other gelatin hydrolysates. As such, TSGH4 was subjected to further fractionation using a Superdex peptide column and two-stage reverse-phase column HPLC chromatography, yielding a subfraction TSGH4-6-2-b, which possessed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity compared with the other fractions. Further LC-ESI/MS/MS analysis of TSGH4-6-2-b suggested two novel peptides (GYDEY and EPGKSGEQGAPGEAGAP), which could have potential as naturally-occurring peptides with antioxidant properties. These promising results suggest that these antioxidant peptides could have applications in food products, nutraceuticals, and cosmetics.