Non-obstructive azoospermia (NOA) is the most severe clinical diagnosis in cases of male infertility. Although in some cases of NOA spermatozoa can be retrieved by microdissection testicular sperm extraction (micro-TESE) to fertilise eggs through intracytoplasmic sperm injection (ICSI), there remains a lack of potential biomarkers for non-invasive diagnosis before micro-TESE surgery. To determine predictive biomarkers for successful sperm retrieval before micro-TESE, the aim of this study was to explore whether microRNAs (miRNAs) were differentially expressed in testicular tissues in NOA patients in whom sperm retrieval had been successful (SSR) versus those in whom it had been unsuccessful (USR) using next-generation small RNA sequencing (RNA-Seq). In all, 180 miRNAs were identified with significantly altered expression levels between SSR and USR testicular tissues. Of these, the expression of 13 miRNAs was upregulated and that of 167 miRNAs was downregulated in the USR compared with SSR group. Unexpectedly, 86 testicular miRNAs were found to be completely absent in the USR group, but showed high expression in the SSR group, suggesting that these miRNAs may serve as biomarkers for micro-TESE and may also play an essential role in spermatogenesis. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that the miRNAs that differed significantly between the USR and SSR groups were involved in cell apoptosis, proliferation and differentiation, which are of considerable importance during spermatogenesis. In summary, this study identified a panel of miRNAs highly expressed in testicular tissues of SSR but not USR NOA patients, providing new insights into specific miRNAs that may play important roles in epigenetic regulation during spermatogenesis. The findings provide a basis for further elucidation of the regulatory role of miRNAs in spermatogenesis and clues to identifying useful biomarkers to predict residual spermatogenic loci in NOA patients during treatment with assisted reproductive technologies.