Background <p>Non-invasive biomarkers are essential for monitoring transplant health, particularly for detecting acute rejection. Donor-derived cell-free DNA (dd-cfDNA) has emerged as a promising biomarker in solid organ transplantation, but optimized methodology and comprehensive evaluation in kidney transplantation (KTx), especially in Japan, are currently limited.</p> Methods <p>We developed a customized approach for dd-cfDNA quantification and evaluated its utility as a biomarker in KTx. In this prospective longitudinal study, 322 blood samples were collected from 39 KTx recipients, mostly within the first year and up to 5 years post-transplant. Quantification was performed using a modified targeted sequencing protocol using a 1,000-SNP probe panel tailored to East Asian genetic backgrounds. The resulting dd-cfDNA values were correlated with clinical parameters and histological findings.</p> Results <p>General longitudinal dynamics of dd-cfDNA in KTx patients were comprehensively described. Elevated dd-cfDNA was consistently associated with acute rejection and graft injuries. In particular, dd-cfDNA significantly distinguished rejection from non-rejection episodes (<i>p</i> = 1.9 × 10^− 4) and showed moderate consistency with serum markers and biopsy findings. Noticeable surges of dd-cfDNA were reported in antibody-mediated rejection and various forms of kidney injury. Distinct from conventional serum markers, dd-cfDNA exhibited a significant inverse correlation with immunosuppressant trough levels (Spearman’s rho = − 0.25, <i>p = </i>8 × 10^− 3).</p> Conclusions <p>Our study demonstrated that dd-cfDNA is a robust, non-invasive biomarker for detecting allograft rejection and injury in KTx and is superior for monitoring patients’ immunosuppressive response. We also present a scalable, cost-effective sequencing-based methodology for quantifying dd-cfDNA, suitable for clinical application.</p>