The development of a chip-based sensor array composed of individually addressable agarose microbeads has been demonstrated for the rapid detection of DNA oligonucleotides. Here, a “plug and play” approach allows for the simple incorporation of various biotinylated DNA capture probes into the bead-microreactors, which are derivatized in each case with avidin docking sites. The DNA capture probe containing microbeads are selectively arranged in micromachined cavities localized on silicon wafers. The microcavities possess trans-wafer openings, which allow for both fluid flow through the microreactors/analysis chambers and optical access to the chemically sensitive microbeads. Collectively, these features allow the identification and quantitation of target DNA analytes to occur in near real time using fluorescence changes that accompany binding of the target sample. The unique three-dimensional microenvironment within the agarose bead and the microfluidics capabilities of the chip structure afford a fully integrated package that fosters rapid analyses of solutions containing complex mixtures of DNA oligomers. These analyses can be completed at room temperature through the use of appropriate hybridization buffers. For applications requiring analysis of ≤102 different DNA sequences, the hybridization times and point mutation selectivity factors exhibited by this bead array method exceed in many respects the operational characteristics of the commonly utilized planar DNA chip technologies. The power and utility of this microbead array DNA detection methodology is demonstrated here for the analysis of fluids containing a variety of similar 18-base oligonucleotides. Hybridization times on the order of minutes with point mutation selectivity factors greater than 10 000 and limit of detection values of ∼10-13 M are obtained readily with this microbead array system.