Lead Zirconate Titanate (PZT)-embedded microcantilevers were fabricated with dimensions of 30 × 90 × 3 μm(3) (width × length × thickness). A thicker PZT layer improved the actuation and enabled long-term data acquisition in common aqueous buffers with a frequency resolution of 20 Hz. A quantitative assay was conducted in the range of 1-20 μM and the resonant frequency was found to increase with the concentration of target DNAs and the probe DNAs were almost saturated at 20 μM. Back-filling with ethyleneglycol-modified alkanethiol was shown to facilitate the hybridization efficiency and stabilize the surface reaction, resulting in a signal enhancement of 40%. We report for the first time how secondary structures in oligonucleotide monolayer change the surface property of a dynamic mode microcantilever and subsequently affect its oscillating behavior. Using fabricated microcantilevers, the real time changes in resonant frequency upon hybridization were measured by utilizing different probe and target sets. The results revealed that the microcantilevers experienced a resonant frequency upshift during the hybridization with complementary DNAs if a dimer structure was present between DNA probes. A resonant frequency downshift was observed for DNA probes that did not contain any complex secondary structures. In addition, the results demonstrate the potential of using these microcantilevers to extract structural information of oligonucleotides.