BACKGROUND: Tuberculosis (TB) is a major global health issue. Early diagnosis of TB is still a challenge. Studies are seeking non-sputum biomarker-based TB test. Emerging evidence indicates potential significance of blood microbiome signatures for diseases. However, blood microbiome RNA profiles are unknown in TB. We aimed to characterize the blood microbiome of TB patients and identify Mycobacterium tuberculosis (Mtb) genome-derived small RNA molecules to serve as diagnostic biomarkers for TB. METHODS: RNA sequencing data of the blood from TB patients and healthy controls were retrieved from the NCBI-SRA database for analyzing the blood microbiome and identifying rRNA-derived small RNA (rsRNA) of Mtb. Small RNA-seq was performed on plasma exosomes from TB patients and healthy controls. The levels of the candidate Mtb rsRNAs were determined by real-time quantitative reverse transcription PCR (RT-qPCR) on plasma from a separate cohort of 73 TB patients and 62 healthy controls. RESULTS: The blood microbiome of TB patients consisted of RNA signals from bacteria, fungi, archaea, and viruses, with bacteria accounting for more than 97% of the total. Reduced blood microbial diversity and abundance of 6 Mycobacterium-associated bacterial genera, including Mycobacterium, Priestia, Nocardioides, Agrobacterium, Bradyrhizobium, and Escherichia, were significantly altered in the blood of TB patients. A diagnostic model for TB based on the 6 genera achieved an area under the curve (AUC) of 0.8945. rsRNAs mapped to the Mtb genome were identified from blood and plasma exosomes of TB patients. RT-qPCR results showed that 2 Mtb-derived rsRNAs, 16 S-L1 and 16 S-L2, could be used as diagnostic biomarkers to differentiate TB patients from healthy controls, with a high co-diagnostic efficacy (AUC = 0.7197). CONCLUSIONS: A panel of blood microbiome signatures and Mtb-derived rsRNAs can serve as blood biomarkers for TB diagnosis.