Circulating miRNAs have been shown to serve as diagnostic/prognostic biomarkers in cancers and other diseases. However, the role of plasma miRNAs in Late-onset hypogonadism (LOH) diagnosis is still unknown. Using Illumina HiSeq2000 sequencing at discovery phase, and then two-step validated by reverse transcriptase polymerase chain reaction (RT-PCR) assays in verification phases. We verified that the expression levels of miR-125a-5p, miR-361-5p and miR-133a-3p were significantly altered in LOH group compared to the control group. The area under the receiver operating characteristic (ROC) curve (AUC) is 0.682, 0.698 and 0.765, respectively. The combination of three miRNAs showed a larger AUC (0.835) that was more efficient for the diagnosis of LOH. Among three miRNAs, miR-133a-3p had the best diagnostic value for LOH with 68.2% sensitivity and 77.3% specificity. Regression analyses show that miR-133a-3p level was negatively associated with the ageing males’ symptoms (AMS) scale. However, miR-361-5p level was positively associated with serum testosterone concentrations. In summary, plasma miRNAs are differentially expressed between LOH and healthy controls. We validated three miRNAs that could act as novel biomarkers for diagnosis of LOH. These miRNAs may be involved in the development of LOH. However, further large and functional studies are warranted to confirm our findings.