With the development of in vitro diagnostics, extracting submicron scale particles from mixed body fluids samples is crucial. In recent years, microfluidic separation has attracted much attention due to its high efficiency, label-free, and inexpensive nature. Among the microfluidic-based separation, the separation based on ultrasonic standing waves has gradually become a powerful tool. A microfluid environment containing a tilted-angle ultrasonic standing surface acoustic wave (taSSAW) field has been widely adapted and designed to separate submicron particles for biochemical applications. This paper investigated submicron particle defection in microfluidics using taSSAWs analytically. Particles with 0.1–1 μm diameters were analyzed under acoustic pressure, flow rate, tilted angle, and SSAW frequency. According to different acoustic radiation forces acting on the particles, the motion of large-diameter particles was more likely to deflect to the direction of the nodal lines. Decreasing the input flow rate or increasing acoustic pressure and acoustic wave frequency can improve particle deflection. The tilted angle can be optimized by analyzing the simulation results. Based on the simulation analysis, we experimentally showed the separation of polystyrene microspheres (100 nm) from the mixed particles and exosomes (30–150 nm) from human plasma. This research results can provide a certain reference for the practical design of bioparticle separation utilizing acoustofluidic devices.