A microfluidic resistive pulse sensing technique offers a simple method for detecting and analysing microparticles in various fields, yet it has challenges such as the noise during detection and low throughput as the signal obtained from a small single sensing aperture and particle position is nonuniform. This study presents a microfluidic chip with multiple detection gates in the main channel to enhance the throughput while maintaining a simple operational system. A hydrodynamic sheathless particle focusing on a detection gate by modulation of the channel structure and measurement circuit with a reference gate to minimize the noise during detection is used for detecting resistive pulses. The proposed microfluidic chip can analyse the physical properties of 200 nm polystyrene particles and exosomes from MDA-MB-231 with high sensitivity with an error of <10% and high-throughput screening of more than 200 000 exosomes per seconds. The proposed microfluidic chip can analyse the physical properties with high sensitivity, so that it can be potentially used for exosome detection in biological and in vitro clinical applications.