MicroRNAs (miRNAs), a subset of small non-coding RNA molecules, play crucial roles in various pathophysiological processes. Studies increasingly indicate that dysregulated miRNAs are associated with bacterial infection. Nevertheless, little is known about miRNAs that respond to infection and their potential clinical value. Our research aimed to identify the serum miRNAs altered during infection.We enrolled serum samples from 73 patients diagnosed with brucellosis and 65 age- and sex-matched control individuals. Illumina sequencing via synthesis (SBS) technology was performed for an initial screen of miRNAs expression profile in serum samples pooled from 29 patients and 29 controls, respectively. A quantitative real-time polymerase chain reaction (qRT-PCR) assay was conducted in the training and validation sets to confirm the concentrations of differentially expressed miRNAs in individual serum samples from 73 patients and 65 controls.The Illumina SBS technology identified 1,372 known miRNAs and 1,893 novel miRNAs in brucellosis patients. The three markedly upregulated miRNAs (miR-15a-3p, miR-7-2-3p, miR-103b) in brucellosis patients were subsequently validated by qRT-PCR assay, of which miR-103b was confirmed to be significantly and steadily increased in the brucellosis patients compared with the controls (>2-fold, P<0.001). The area under the receiver operating characteristic (ROC) curve (AUC) for miR-103b was 0.714 (95% CI, 0.624-0.804). Bioinformatics analysis predicted that some putative target genes of miR-103b are involved in immune regulation or the processes of apoptosis and autophagy in humans.The serum miR-103b level markedly increases after infection and has the potential to serve as an auxiliary diagnostic indicator for infection that deserves further investigation.