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A cytosolic medium-chain alcohol dehydrogenase Gox0313 from Gluconobacter oxydans DSM2003 was heterologously expressed in Escherichia coli BL21 (DE3) and the resulting proteins were purified and characterized. The recombinant enzyme was confirmed to have a good ability to selectively oxidize the terminal hydroxyl group of varied aliphatic and aromatic diols to the corresponding hydroxyl aldehydes, with no oxidative activities toward hydroxyl aldehydes in the presence of NAD+. This enzyme could not oxidize sec-alcohols. Among the primary diols, ethylene glycol was efficiently converted to glycolaldehyde by Gox0313 coupled with NADH oxidase-2 (NOX-2) from Lactobacillus brevis ATCC367 for NAD+ regeneration. Under the optimal conditions using this enzyme-coupled system (1 U/mL Gox0313 and 4 U/mL NOX-2), 42.32 mM glycolaldehyde was produced from 50 mM ethylene glycol in 8 h with a yield of 83.2%. When the enzyme concentration of Gox0313 was increased to 120 U/mL in the reaction, 500 mM of ethylene glycol was converted to 484.2 mM of glycolaldehyde in 14 h, resulting in a yield of 96.8%. With respect to the superior catalytic activity to primary diols, Gox0313 might be a potentially promising biocatalyst for bioproduction of glycolaldehyde and other hydroxyl aldehydes.