A highly sensitive electrogenerated chemiluminescence (ECL) method was developed for trace analysis of 5-hydroxymethylcytosine double-stranded DNA (5-hmC-dsDNA). The poly-(dimethyldiallyl ammonium chloride)/multiwalled carbon nanotubes composite was assembled on a bare glassy carbon electrode (GCE) to provide high specific surface area on which the loadable capacity of 5-hmC-dsDNA and 5-hmC antibody can be greatly increased. The derivative of ruthenium (II) bibyridine, Ru (bpy) (dcbpy)NHS, coupled with 5-hmC antibody to activate an ECL reaction when the applied potential was biased at 1.4 V vs. Ag/AgCl. The loading ratio of substrates were optimized to enhance the detection sensitivity of 5-hmC-dsDNA. It was found that the ECL intensity was a piecewise linear function of the concentration of 5-hmC-dsDNA over the range of 1.0 × 10-2.0 × 10 M. A linear relationship of I = 6850.3 C + 863.8 (R = 0.9954) was obtained from 0.01 to 0.2 nM, while the fitting equation of I = 3840.0 C + 1392.4 (R = 0.9974) is for the concentration range of 0.2 - 2.0 nM. The detectable low limit can reach to 2.3 × 10 M. Formation of the antigen-antibody immunocomplex in highly concentrated solutions should undertake most of the responsibility for a decrease in slope. Furthermore, reliability, reproducibility and practicability of the ECL method have been proved to perform well, even in real bio-tissues, suggesting promising prospect in early diagnosis of cancer.