This study addresses the growing interest in developing new acoustophoresis designs for efficient particle separation, introducing a novel concave–convex electrode design for lymphocyte separation. Initially, a numerical model for acoustophoresis was employed and validated against existing experimental results in the literature with a 4% variance, based on the finite element method. Furthermore, in order to ensure the accuracy of the performed simulations, a mesh independency approach was employed for the piezoelectric substrate, alongside an investigation into resonant frequencies across the computational domain. These analyses were conducted to ensure that the results approximate experimental findings more closely and identify the frequency at which the maximum surface displacement occurs, making the results empirically reliable. As a major innovation, a new concentric concave–convex electrode design was introduced, and then the separation distance of targeted particles, as the goal parameter, was studied relative to the geometrical design and acoustofluidic operation parameters of the microfluidic chip. Through numerical analysis, the flow rate ranging from 7 to 14 μl/min and the applied radio frequency signal amplitude ranging from 16 to 26 V were investigated simultaneously. Results demonstrated the microfluidic chip's capability to function effectively across the entire range of voltage and flow rates examined. At the chip's highest operational point, with a flow rate of 13 μl/min and an applied radio frequency signal amplitude of 24 V, particle separation distance reached up to 380 μm. Under similar flow rates, cell conditions, and microchannel length, the particle separation distance has been improved by about 26% as compared with the standard electrode pattern, revealing a significant enhancement in separation efficiency and output purity. Moreover, due to the predominantly radial propagation of the acoustic waves and the expanding acoustic aperture, the resultant standing wave pattern spans a greater length of the microchannel. Assuming a constant injection velocity, this consequently extends the effective exposure time of particles to the acoustic radiation force, allowing for an increase in Stokes drag force. Given that drag force increases with velocity, it enables the opportunity to introduce higher input flow rates and throughput.