Objective <p>Pulmonary tuberculosis (PTB) remains a significant global health problem in the 21st century. This study explored the diagnostic value of cell-free DNA (cfDNA) technology-assisted acid-fast staining (AFS) for PTB.</p> Methods <p>A retrospective analysis was conducted on 237 suspected PTB patients. All sectioned specimens underwent AFS, and plasma samples were tested for cfDNA. Patients were categorized into positive and negative groups based on etiology and clinical symptom reference standards. The diagnostic value of cfDNA-assisted AFS was analyzed using receiver operating characteristic (ROC) curves. The concordance rates of cfDNA and AFS were calculated based on true-positive and true-negative cases, and their consistency was assessed using the Kappa test.</p> Results <p>Under the etiological and clinical reference standards, the true-positive detection rate for PTB of cfDNA was higher than that of AFS (79.05% vs. 65.87% and 68.72% vs. 55.38%, respectively). cfDNA-assisted AFS exhibited superior diagnostic performance over either test alone, yielding higher AUCs (0.946 vs. 0.808 for AFS and 0.874 for cfDNA under etiological reference standard; 0.853 vs. 0.717 for AFS and 0.832 for cfDNA under clinical reference standard). Similarly, the combined approach significantly enhanced sensitivity (96.41% and 82.56%) and specificity (92.86% and 88.10%). cfDNA-assisted AFS showed higher Kappa values than either AFS or cfDNA alone under both reference standards (etiology: κ = 0.889 vs. κ = 0.503 vs. κ = 0.662; clinical symptom: κ = 0.556 vs. κ = 0.246 vs. κ = 0.425).</p> Conclusion <p>CfDNA-assisted AFS enhances the diagnostic performance for PTB, providing a novel auxiliary tool for the clinical diagnosis of PTB.</p>