A new α-neoagarobiose hydrolase (NABH) called AgaWH117 was cloned from Agarivorans gilvus WH0801. The gene encoding this hydrolase consists of 1,086 bp and encodes a protein containing 361 amino acids. This new NABH showed 74% amino acid sequence identity with other known NABHs. The molecular mass of the recombinant AgaWH117 was estimated to be 41 kDa. Purified AgaWH117 showed endolytic activity during neoagarobiose degradation, yielding 3,6-anhydro-l-galactose (l-AHG) and d-galactose as products. It showed a maximum activity at a temperature of 30 °C and a pH of 6.0 and was stable at temperatures below 30 °C. Its Km and Vmax values were 2.094 mg/mL and 6.982 U/mg, respectively. The cloning strategy used and AgaWH117 isolated in this study will provide information on the saccharification process of marine biomass. This study provides a method to produce l-AHG from agarose by using AgaWH117 without an acid and describes its one-step purification by using Bio-Gel P2 chromatography.