Edible bird’s nest (EBN) hydrolysates have been proven to exhibit enhanced bioactivities. However, being a macromolecule, fractions with different molecular weights would have different properties and bioactivities. Hence, this research was aimed to determine the chemical properties and antioxidant activities of freeze-dried (EBNFD) and spray-dried EBN (EBNSD) hydrolysates fractionated using gel permeation chromatography (GPC). Overall, two well-separated fractions were identified (EBNfFD1, EBNfFD2, EBNfSD1 and EBNfSD2). EBNFD demonstrated significantly higher (P ≤ 0.05) peptide (3.6%), total carbohydrate (27.7%) and sialic acid (18.2%) contents than that of EBNSD. Similar trend was observed in low molecular weight fractionates (EBNfFD2 and EBNfSD2). Meanwhile, the first fractionates (EBNfFD1 and EBNfSD1) exhibited significantly higher (P ≤ 0.05) hydroxyl radical (•OH) scavenging activity. Fourier transform infrared (FTIR) spectroscopy demonstrated that all EBN fractionates have similar spectrum, except in the region of N—H (amide II) and C—H alkyl group. In conclusion, EBN fractionates with different molecular weights showed different chemical properties and antioxidant activities.