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Background: Atopic dermatitis (AD) is a prevalent inflammatory skin disease. Ample evidence has shown that non-coding RNAs play important roles in the progression of AD, however, the function of plasma microRNAs in AD is poorly understood. Objectives: To identify key plasma microRNAs and explore their potential roles in AD. Materials and Methods: Plasma microRNAs from five children with AD and five control children were sequenced by microRNA sequencing (miRNA-seq) and five differentially expressed microRNAs were verified by RT-qPCR in 30 AD and 15 control children. The most differentially expressed microRNA, hsa-miR-194-5p, was selected for further analysis. Human epidermal keratinocytes were subjected to RNA sequencing following over-expression of hsa-miR-194-5p, and down-regulated genes were detected by RT-qPCR. The diagnostic potential of hsa-miR-194-5p in AD was evaluated based on receiver operating characteristic (ROC) curve analysis. Further hsa-miR-194-5p-regulated expression and gene-hsa-miR-194-5p interactions were evaluated by western blotting and luciferase assays, respectively. Results: We identified 40 differentially expressed microRNAs, 26 up-regulated and 14 down-regulated, in children with AD compared with controls. Among the five verified plasma microRNAs, the most significant change was down-regulated hsa-miR-194-5p, which was shown to potentially serve as a biomarker for AD based on ROC analysis. Twenty-two down-regulated genes were observed following hsa-miR-194-5p over-expression, which were significantly associated with keratinocyte differentiation and establishment of the skin barrier. Moreover, HS3ST2protein expression was down-regulated following over-expression of hsa-miR-194-5p, and the 3′-UTR of HS3ST2 was shown to bind to hsa-miR-194-5p. Conclusion: Hsa-miR-194-5p might be involved in the pathogenesis of AD by regulating HS3ST2 expression.