Immune rejection limits long-term renal allograft survival, yet current diagnostics lack non-invasive, and precise detection. Here, donor-derived circulating podocytes (CPCs) are identified as a blood-based surrogate of acute rejection. A magnetically reversible herringbone microfluidic chip equipped with epithelial cell adhesion molecule (EpCAM) antibody-functionalized magnetic beads efficiently isolates CPCs from 1 mL of peripheral blood (capture efficiency 84.4%, release 95.5%, viability 96.0%). Among 65 participants, only transplant recipients with biopsy-confirmed rejection exhibited≥35 CPCs mL^- 1 (AUC = 1.0). Fluorescence in situ hybridization (FISH) of sex-mismatched transplants confirmed donor origin. Single-cell RNA-seq of 10 isolated CPCs revealed up-regulation of podocyte-injury and innate/adaptive immune genes (such as NF-κB, TNF, NOD-like receptor pathways) in rejection versus stable samples. CPC enumeration thus provides a minimally invasive, mechanistically informed warning of renal allograft rejection.