We developed a novel electrogenerated chemiluminescence (ECL) biosensing method for quantitative detection of DNA hydroxymethylation (5-hydroxymethylcytosine, 5-hmC) using T4 beta-glucosyltransferase (beta-GT) and graphene oxide (GO) for enzymatic and chemical modification of the 5-hmC. beta-GT selectively glucosylated the hydroxymethyl group of 5-lnC to improve selectivity, whereas GO, which loads substantial amounts of the ECL reagent, was used as the signal amplification element to enhance assay sensitivity. The ECL bioassay involved the capture of DNA containing 5-hmC, which was chemically modified with a glucose moiety, through hybridization with complimentary 5'-thiol-modified DNA on a glassy carbon electrode (GCE) under the catalysis of beta-GT. The dichlorotris (1,10-phenanthroline) ruthenium -assembled GO (Ru(phen)(3)(2+)/GO) composite was coupled to the electrode surface via a bridging agent, and provided excellent ECL signal. The bridging agent used was 3-aminophenylboronic acid (APBA), boronic acid and its derivatives can interact with 1,2-or 1,3-diols (such as glucose) to generate 5-or 6 -membered cyclic boronate esters. The obtained ECL intensity was positively correlated with the concentration of DNA containing 5-hmC, the linear range was from 0.01 pM to 1.00 nM, with a low limit of detection of 3.84 fM. Our results demonstrate that use of enzymatic and chemical modification in combination with highly sensitive ECL is a facile and promising approach for DNA hydroxymethylation analysis.