We report a temperature-controlled microfluidic acoustophoresis device capable of separating particles and transferring blood cells from undiluted whole human blood at a volume throughput greater than 1 L h−1. The device is fabricated from glass substrates and polymer sheets in microscope-slide format using low-cost, rapid-prototyping techniques. This high-throughput acoustophoresis chip (HTAC) utilizes a temperature-stabilized, standing ultrasonic wave, which imposes differential acoustic radiation forces that can separate particles according to size, density and compressibility. The device proved capable of separating a mixture of 10- and 2-μm-diameter polystyrene beads with a sorting efficiency of 0.8 at a flow rate of 1 L h−1. As a first step toward biological applications, the HTAC was also tested in processing whole human blood and proved capable of transferring blood cells from undiluted whole human blood with an efficiency of 0.95 at 1 L h−1 and 0.82 at 2 L h−1.