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Sea cucumber-derived peptide, a potential candidate for combating aging, is currently under-developed. In the present study, we reported the development of a green approach to efficiently prepare a high-value anti-aging oligopeptide fraction with desired color and low viscosity from a low-edible-value sea cucumber (Stichopus variegates) containing highly viscous polysaccharide using enzymatic hydrolysis, acid-heat treatment and ultrafiltration. The analysis by UPLC-MS/MS revealed the peptide composition characteristic of S. variegates peptide fraction, suggesting the acceptable stabilities of oligopeptides during citric acid-heat processing. In vivo studies demonstrated that S. variegates peptide fraction effectively promoted longevity in normal and D-galactose-induced aging fruit flies and attenuated D-galactose-induced oxidative injury in mice via up-regulating Klotho expression, activating SOD and GSH-Px, and inhibiting lipid peroxidation and protein oxidation. Our results also validated the stability of longevity promoting efficacy and anti-oxidative stress action of S. variegates peptide after citric acid-heat treatment.