The study introduces a DNA framework signal amplification platform (DSAP) for high-throughput systemic immune monitoring. DSAP is designed to overcome the limitations of conventional flow cytometry, which is time-consuming, technically sensitive, and costly, especially for large-scale sample testing. The DSAP platform uses post-systematic evolution of ligands by exponential enrichment and DNA tetrahedral framework-structured probe design to achieve high-sensitive detection of diverse immune cells, including CD4+, CD8+ T-lymphocytes, and monocytes, with a detection limit as low as 1/100 μl. The DSAP system can perform one-step immune-cell phenotyping in 30 minutes without cell washing or subset analysis, showing comparable accuracy to flow cytometry while significantly reducing detection time and cost. As a proof-of-concept, DSAP demonstrated excellent diagnostic accuracy in staging immunodeficiency in 107 HIV patients (AUC > 0.97) within 30 minutes, making it suitable for HIV infection monitoring and screening. The study also highlights the potential of DSAP in rapid clinical immune monitoring and the development of point-of-care devices.The study introduces a DNA framework signal amplification platform (DSAP) for high-throughput systemic immune monitoring. DSAP is designed to overcome the limitations of conventional flow cytometry, which is time-consuming, technically sensitive, and costly, especially for large-scale sample testing. The DSAP platform uses post-systematic evolution of ligands by exponential enrichment and DNA tetrahedral framework-structured probe design to achieve high-sensitive detection of diverse immune cells, including CD4+, CD8+ T-lymphocytes, and monocytes, with a detection limit as low as 1/100 μl. The DSAP system can perform one-step immune-cell phenotyping in 30 minutes without cell washing or subset analysis, showing comparable accuracy to flow cytometry while significantly reducing detection time and cost. As a proof-of-concept, DSAP demonstrated excellent diagnostic accuracy in staging immunodeficiency in 107 HIV patients (AUC > 0.97) within 30 minutes, making it suitable for HIV infection monitoring and screening. The study also highlights the potential of DSAP in rapid clinical immune monitoring and the development of point-of-care devices.