Water-soluble squid melanin fractions were firstly prepared using ultrasound-assistant degradation method under alkaline condition, which is optimized by response surface methodology. The processing melanin fractions were divided into different molecular weight (Mw) fractions by membrane separation (below 10 kDa, among 10-50 kDa and over 50 kDa). The AFM image and particle-size analysis showed monomer units of the melanin were destroyed, and huge polymers were degraded into smaller soluble particles after ultrasound. While, UV, IR and solid (13)C NMR spectra indicated that the basic structure of melanin fraction was still retained after ultrasound process. Further analysis showed soluble melanin fractions obtained in 0.5 and 1 M NaOH, with Mw above 10 kDa exhibited much higher in vitro antioxidant potency. The IC50 of these fractions (IC50 among 19-80 μg) on scavenging O 2 ∙¯ is more efficient than carnosine (IC50 = 355 μg/ml.), a commercialized antioxidant. They (IC50 mong 115-180 μg/ml) are as efficient as carnosine (IC50 = 110 μg/ml) on scavenging ∙OH. Our research has reported a novel method for preparation of water-soluble melanin fractions from squid ink, which could be a promising free radical scavenger from nature resource.