A DNA framework signal amplification platform (DSAP) was developed for high-throughput immune monitoring. This platform uses post-SELEX aptamer optimization and DNA tetrahedral framework (DTF)-structured HCR probes to achieve high-sensitive detection of immune cells, including CD4+, CD8+ T-lymphocytes, and monocytes. DSAP enables rapid, one-step immune-cell phenotyping without cell washing or subset analysis, achieving results comparable to flow cytometry but with significantly reduced time and cost. As a proof-of-concept, DSAP demonstrated excellent diagnostic accuracy in immunodeficiency staging for 107 HIV patients (AUC > 0.97) within 30 minutes, showing potential for HIV infection monitoring and screening. The DSAP platform was constructed by optimizing aptamers through post-SELEX and designing DTF-structured HCR probes. This approach enhances signal amplification and improves detection sensitivity. The DSAP platform was validated for its sensitivity, specificity, and structural stability, with results showing strong linear correlations between MFI and cell concentrations. The platform was tested on various immune cell types, including CD4+ T, CD8+ T lymphocytes, and monocytes, demonstrating high accuracy and reliability. In clinical applications, DSAP showed robust linear correlations between signal-to-noise ratios and FCA results, with an R² coefficient of 0.959. The platform was also tested on blood samples from patients with different diseases, showing consistent results across various immune statuses. DSAP demonstrated high accuracy in diagnosing immunodeficiency stages in HIV patients, with AUC values exceeding 0.97 for different stages. Additionally, DSAP was applied to cancer patients at different stages, showing potential for immune monitoring and treatment planning. The DSAP platform offers a promising solution for rapid immune monitoring and has the potential to enhance the development of point-of-care devices. Its high sensitivity, specificity, and ability to detect a wide range of immune cells make it a valuable tool for clinical applications. The platform's ability to provide accurate and reliable results in a short time makes it suitable for large-scale health screenings and clinical diagnostics.A DNA framework signal amplification platform (DSAP) was developed for high-throughput immune monitoring. This platform uses post-SELEX aptamer optimization and DNA tetrahedral framework (DTF)-structured HCR probes to achieve high-sensitive detection of immune cells, including CD4+, CD8+ T-lymphocytes, and monocytes. DSAP enables rapid, one-step immune-cell phenotyping without cell washing or subset analysis, achieving results comparable to flow cytometry but with significantly reduced time and cost. As a proof-of-concept, DSAP demonstrated excellent diagnostic accuracy in immunodeficiency staging for 107 HIV patients (AUC > 0.97) within 30 minutes, showing potential for HIV infection monitoring and screening. The DSAP platform was constructed by optimizing aptamers through post-SELEX and designing DTF-structured HCR probes. This approach enhances signal amplification and improves detection sensitivity. The DSAP platform was validated for its sensitivity, specificity, and structural stability, with results showing strong linear correlations between MFI and cell concentrations. The platform was tested on various immune cell types, including CD4+ T, CD8+ T lymphocytes, and monocytes, demonstrating high accuracy and reliability. In clinical applications, DSAP showed robust linear correlations between signal-to-noise ratios and FCA results, with an R² coefficient of 0.959. The platform was also tested on blood samples from patients with different diseases, showing consistent results across various immune statuses. DSAP demonstrated high accuracy in diagnosing immunodeficiency stages in HIV patients, with AUC values exceeding 0.97 for different stages. Additionally, DSAP was applied to cancer patients at different stages, showing potential for immune monitoring and treatment planning. The DSAP platform offers a promising solution for rapid immune monitoring and has the potential to enhance the development of point-of-care devices. Its high sensitivity, specificity, and ability to detect a wide range of immune cells make it a valuable tool for clinical applications. The platform's ability to provide accurate and reliable results in a short time makes it suitable for large-scale health screenings and clinical diagnostics.