A hemolysis‐free and highly efficient plasma separation platform enabled by enhanced diamagnetic repulsion of blood cells in undiluted whole blood is reported. Complete removal of blood cells from blood plasma is achieved by supplementing blood with superparamagnetic iron oxide nanoparticles (SPIONs), which turns the blood plasma into a paramagnetic condition, and thus, all blood cells are repelled by magnets. The blood plasma is successfully collected from 4 mL of blood at flow rates up to 100 µL min−1 without losing plasma proteins, platelets, or exosomes with 83.3±1.64% of plasma volume recovery, which is superior over the conventional microfluidic methods. The theoretical model elucidates the diamagnetic repulsion of blood cells considering hematocrit‐dependent viscosity, which allows to determine a range of optimal flow rates to harvest platelet‐rich plasma and platelet‐free plasma. For clinical validations, it is demonstrated that the method enables the greater recovery of bacterial DNA from the infected blood than centrifugation and the immunoassay in whole blood without prior plasma separation.

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