We present a multiplex microarray-based assay of DNA fragments, which allows the detection of less than 10000 DNA fragments in a sample of 100 μL (corresponding to ∼0.1 fM analyte concentration) in less than 5 min. High speed and sensitivity are due to three main features of the assay. First, biotinylated adapter oligonucleotides are hybridized to the DNA fragment. Second, it is electrophoretically concentrated from the sample onto the microarray. Third, biotin labels are detected by scanning the microarray surface with streptavidin-coated magnetic beads. Prior to analysis, dsDNA fragments and genomic DNA samples were first denatured and then annealed in the presence of blocking oligonucleotides, generating ssDNA fragments capable of hybridizing with oligonucleotide probes on the microarray. The multiplexity of the assay system was demonstrated by the simultaneous detection of the genomic DNAs of three microorganisms: E. coli, B. cereus, and M. neoaurum.