The gonad is one of the major byproducts of sea cucumber. Four novel antioxidant peptides (NPWGQ, PGHPF, VPYPR and ATGPQGPAGQRGPAGPTGPTGPAG) were isolated and identified from sea cucumber gonad proteins through enzymatic hydrolysis and antioxidant activity-guided fractionation, bioinformatics approaches and in silico screening. These peptides demonstrated great free radical (2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS))-scavenging activity and notable superoxide dismutase (SOD)-activation capacity. Molecular docking and molecular dynamics simulation data suggested that these peptides could form strong binding with SOD through hydrogen bonding, electrostatic interactions, and hydrophobic interactions. Among these peptides, NPWGQ displayed the most potent antioxidant and SOD-activating effects. Through searching known databases, these peptides did not show potential toxicity and are generally considered safe. The present study provides crucial theoretical support for comprehensively utilizing sea cucumber (Holothuroidea) gonad by-products and generating high-value functional food ingredients or dietary supplements.