Abstract: This study investigated the anti-aging effects of three ultrafiltration fractions (EPU-1 (<5 kDa), EPU-2 (5–8 kDa), and EPU-3 (>8 kDa)) derived from the enzymatic hydrolysate of Perinereis aibuhitensis on Caenorhabditis elegans. The basic nutritional composition of each fraction was determined, and their in vitro antioxidant activity was evaluated based on hydroxyl radical and superoxide anion scavenging capacities. Using nematode as the model organism, the effects of these fractions were assessed with respect to stress resistance, lifespan, locomotion, fecundity, and lipofuscin accumulation. Furthermore, the expression levels of proteins associated with the insulin/IGF-1 signaling (IIS) pathway were measured to explore the underlying anti-aging mechanisms. The results indicated that EPU-1 and EPU-2 were rich in peptides, with contents of 44.20% and 40.41%, respectively, whereas EPU-3 contained only 4.60% peptides. All three fractions exhibited strong in vitro and in vivo antioxidant activities and significantly delayed aging in nematode. Among them, EPU-1 demonstrated the most potent antioxidant and anti-aging effects. Its hydroxyl radical and superoxide anion scavenging capacities were significantly higher than those of EPU-2 and EPU-3 (P<0.05). Compared with the control group, EPU-1 significantly enhanced the nematode's resistance to acute oxidative stress (P<0.05) and thermal stress (P<0.05), extended lifespan, and increased brood size (P<0.05). All three fractions significantly improved locomotor capacity and reduced lipofuscin accumulation (P<0.05). Moreover, EPU-1 significantly downregulated the expression of daf-2 and age-1 (P<0.01) and upregulated daf-16 and skn-1 (P<0.05). In conclusion, the ultrafiltration fractions of Perinereis aibuhitensis enzymatic hydrolysate possess notable antioxidant and anti-aging properties, with EPU-1 exhibiting the strongest efficacy. These findings provide a scientific basis for the potential application of these fractions in functional foods and related health products.