Abstract: 【Objective】 This study aimed to identify the miRNAs in Lycium barbarum L. fruits and analyze their regulation roles during fruit development， providing theoretical basis for cultivation of superior varieties and formation of high-quality fruits of Lycium barbarum L. in the future. 【Method】 Lycium barbarum L. fruits at three developmental stages （green fruit stage， color turning stage， and maturity stage） were collected as materials. The Illumina HiSeq 2500 platform was used to perform miRNA sequencing， combined with bioinformatic methods to identify potential miRNAs. By compa-ring the expression of miRNAs of Lycium barbarum L. fruits at different developmental stages， differentially expressed miRNAs at the three developmental stages were screened using analysis of variance. GSTAr （v1.0） was employed to predict target genes of differentially expressed miRNAs and perform GO functional annotation and KEGG pathway enrichment analysis. The real-time fluorescence quantitative PCR was used for validation. 【Result】 A total of 1013 miRNAs were identified from the Lycium barbarum L. fruits， including 51 known miRNAs， 386 conservative miRNAs， and 576 novel miRNAs， with the number of miRNAs of 24 nt was the highest. Conservative analysis indicated that Lycium barbarum L. shared the largest number of highly similar miRNAs with soybean. A total of 213 differentially expressed miRNAs were screened， and 848 target genes were predicted. GO functional annotation analysis showed that the target genes were mainly enriched in terms including regulation of DNA-templated transcription， defense response， nucleus， cytoplasm， protein binding， and DNA-binding transcription factor activity. KEGG signaling pathway enrichment analysis revealed that target genes were significantly enriched in pathways including plant-pathogen interaction， plant hormone signal transduction， and carotenoid biosynthesis. A miRNA-target gene-KEGG signaling pathway regulatory model was established， and identify some important miRNA-target gene modules that performed multiple biological regulatory functions were identified. Real-time fluorescence quantitative PCR validation confirmed the reliability of the miRNA sequencing data. 【Conclusion】 miRNAs and their target genes play important regulation roles in the development of Lycium barbarum L. fruits， suggesting that miRNA-target gene modules such as miR156-SPL， miR167-ARF， miR159-MYB， and miR482-bHLH exert important functions in the growth and development， stress response， and secondary metabolite biosynthesis of Lycium barbarum L. fruits.