The RPS (Resistive Pulse Sensing) technique is a popular tool for the label-free detection of particles. This paper describes a simple, cost-effective PDMS nanofluidic chip for the detection and characterization of nanoparticles based on the differential RPS technique with high resolution and sensitivity. The chip is composed of two layers of PDMS slabs. Microchannel systems fabricated by the photolithography method on the top layer are used for sample loading and differential signal acquisition, and a straight nanochannel on the bottom layer fabricated by an unconventional approach bridging the gap between the microchannels works as an RPS sensing gate. A single-stage differential amplifier is used to amplify the RPS signals when particles or DNA pass through the sensing gate. It was demonstrated that this nanofluidic RPS chip can detect nanoparticles as small as 23 nm with a high SNR (Signal-to-Noise Ratio). The experimental results also show that the device is able to distinguish nanoparticles of smaller size differences such as 60 nm and 83 nm with high resolution, showing superior performance in comparison with the results obtained from DLS (Dynamic Light Scattering). This differential nano-RPS chip was also applied to detect the translocation of dsDNA molecules.