This paper reports an IC-compatible method for fabricating a PDMS-based resistive pulse sensing (RPS) device with embedded nanochannel (nanochannel-RPS) for label-free analysis of biomolecules and bionanoparticles, such as plasmid DNAs and exosomes. Here, a multilayer lithography process was proposed to fabricate the PDMS mold for the microfluidic device comprising a bridging nanochannel, as the sensing gate. RPS was performance by placing the sensing and excitation electrodes symmetrically upstream and downstream the sensing gate. In order to reduce the noise level, a reference electrode was designed and placed beside the excitation electrode. To demonstrate the feasibility of the proposed nanochannel-RPS device and sensing system, polystyrene micro- and nanoparticles with diameters of 1 μm and 300 nm were tested by the proposed device with signal-to-noise ratios (SNR) ranging 9.1-30.5 and 2.2-5.9, respectively. Furthermore, nanochannel with the height of 300 nm was applied for 4 kb plasmid DNA detection, implying the potential of the proposed method for label-free quantification of nanoscale biomolecules. Moreover, HeLa cell exosomes, known as a well-studied subtype of extracellular vesicles, were measured and analyzed by their size distribution. The result of the resistive pulse amplitude was well corresponding to the that of nanoparticle tracking analysis (NTA). The proposed nanochannel-RPS device and the sensing strategy are not only capable for label-free analysis for nanoscale biomolecules and bionanoparticle, but also cost-effective for large-scale manufacturing.