In this study, a new and sensitive signal-off electrogenerated chemiluminescence (ECL) biosensing method for the quantification of 5-hydroxymethylcytosine in DNA (5-hmC-DNA) was developed. The method achieved simple and sensitive detection of 5-hmC-DNA based on the glycosylation of 5-hmC, combining both the amplification function of gold nanoparticles (AuNPs) and the high quenching efficiency of the tris(2, 2'-ripyridine) dichlororuthenium(II) (Ru(bpy))-ferrocene (Fc) system. First, the electrode modified with a mixture of Nafion and AuNPs was utilized as the platform for electrostatically adsorbing Ru(bpy)(an ECL-emitting species) and assembling 5-hmC-DNA. The 5-hmC-DNA was glycosylated by T4 β-glucosyltransferase, yielding β-glucosyl-5-hydroxymethyl-cytosine in DNA (5-ghmC-DNA). Finally, quencher-FcBA was further covalently bound to 5-ghmC-DNA through formation of boronate ester covalent bonds between boronic acid and cis-diols of 5-ghmC, resulting in a decrease in ECL intensity. The results indicated that the decreased ECL intensity was directly linear to the concentration of 5-hmC-DNA in the range from 1.0×10 to 5.0×10M with a low detection limit of 1.63×10M. In addition, this ECL method was demonstrated to be useful for the quantification of 5-hmC in clinical serum samples. Moreover, the method allowed good discrimination among cytosine (5-C), 5-methylcytosine (5-mC), and 5-hmC in DNA.