In this work, a streaming potential analyzer was constructed based on a composite PDMS-on-glass microchannel and was applied to the label-free DNA detection. Peptide nucleic acid (PNA) probes were covalently attached to the glass substrate to facilitate hybridization with the target DNA at low ionic strength and provide minimized baseline signal for streaming potential analysis. The hybridization of the neutral PNA in the microchannel with the target DNA produced a negatively charged complex, which caused the change of zeta potential and in turn the measured streaming potential. The change of zeta potential was dependent on the hybridization time as well as the concentration and length of the target DNA, through which the complementary target DNA was distinguished from non-complementary DNA with a detection limit of 10 nM. The PNA probes could be recovered in situ and reused without significant capacity loss. The change of the zeta potential was decreased by only 4% after three cycles of PNA-DNA hybridization and PNA recovery. The microfluidic streaming potential analyzer functionalized with PNA probes provides a simple, inexpensive and reusable platform for nucleic acid detection.