In this paper, a novel microfluidic device for sheathless particle focusing and separation in viscoelastic fluid is proposed. The device consists of two stages: a straight channel section with asymmetrical expansion–contraction cavity arrays (ECCA section) for sheathless Dean-flow-coupled elasto-inertial particle focusing (1st stage), and a straight channel section for viscoelastic particle separation (2nd stage). In stage 1, particles with diameters of 5 μm and 13 μm were both focused at the opposite sides of the cavities. Then, the particles were subsequently separated at the 2nd stage based on the differential focusing dependency on size. The effects of flow rates and channel length on particle separation were investigated. Particle separation in both viscoelastic fluid and Newtonian fluid was also compared to elucidate the differences. In addition, particle separation in the straight channel and integrated ECCA straight channel was also studied. The proposed device was used to separate human Jurkat cells (an immortalized T cell line) and yeast cells. Experimental results show that this technique offers an efficient, continuous, and sheathless particle separation in viscoelastic fluid.