An ultrasensitive DNA hybridization detection method based on electrogenerated chemiluminescence (ECL) using polystyrene microspheres/beads (PSB) as the carrier of the ECL labels, namely, tris(2,2'-bipyridyl)ruthenium(II) tetrakis(pentafluorophenyl)borate (Ru(bpy)3[B(C6F5)4]2), is reported. Probe single-stranded DNA (p-ssDNA) was attached to the surface of magnetic beads (MB) and hybridized with target-ssDNA (t-ssDNA) with immobilized PSB containing a large number of water insoluble Ru(bpy)3[B(C6F5)4]2 species (approximately 7.5 x 10(9) molecules/bead). With this approach a large amplification factor of Ru(bpy)3[B(C6F5)4]2 molecules for each t-ssDNA can be achieved, when each PSB is attached to a limited number of t-ssDNA. The p-ssDNA-MB <--> t-ssDNA-PSB/Ru(bpy)3(2+) conjugates formed were magnetically separated from the reaction media and dissolved in MeCN containing tri-n-propylamine (TPrA) as an ECL coreactant. ECL was produced with a potential scan from 0 to 3.0 V versus Ag/Ag+, and the integrated ECL intensity was found to be linearly proportional to the t-ssDNA concentration in a range of 1.0 fM to 10 nM under optimized conditions. ECL signals associated with two base pair mismatched ssDNA and noncomplementary ssDNA can be distinguished well from the ECL signal related to the complementary DNA hybridization. A Poisson distribution is followed when a large number of MB reacts with PSB, and the minimum number of 1.0- and 2.8-microm diameter MB required to bind and magnetically separate a single 10-microm diameter PSB from the reaction solution was estimated to be three and one, respectively. The principle described in this paper could be also applied to many other ECL analyses, such as immunoassays.