Background The treatment of Traditional Chinese Medicine (TCM) in recurrent aphthous stomatitis (RAS) based on syndrome differentiation has won the international acceptance, but the molecular mechanism of excess-heat syndrome and yin-deficiency syndrome of RAS remains unclear. Objective To clarify specific microRNAs (miRNAs) and their functions in RAS patients with excess-heat or yin-deficiency. Methods Serum samples were collected from patients meeting the RAS diagnostic criteria of excess-heat or yin-deficiency syndrome and healthy individuals. Core miRNAs were then identified under miRNA microarray analyses. Target prediction and bioinformatic analyses were carried out and gene-pathway-networks were visualized to better understand the relationship between different genes and pathways. Results (1) 90 individuals meeting the inclusion criteria were collected in this study. Among them, 9 miRNAs were screened out in excess-heat syndrome group (EH group), with 1 upregulated and 8 downregulated. And four miRNAs (hsa-miR-20b–5p, hsa-miR-122–5p, hsa-miR-483–5p and hsa-miR-3197) were validated by real-time PCR method. 14 miRNAs were screened out in yin-deficiency syndrome group (YD group) (7 upregulated and 7 downregulated). And hsa-miR-17–5p, hsa-miR-106–5p and hsa-miR-20b–5p were validated. (2) A total of 4,776 target genes were identified in EH group which enriched in GO categories including nervous system development and calcium ion binding and pathway such as PI3K-Akt signaling pathway and Calcium signaling pathway. 10,172 target genes were identified in YD group which enriched GO categories included protein binding and positive regulation of transcription from RNA polymerase II promoter and pathway included MAPK signaling pathway and Ras signaling pathway. Conclusion Hsa-miR-20b–5p in patients with RAS could act as the novel target for the classification of the syndrome. It is upregulated in RAS patients with excess-heat syndrome while downregulated in patients with yin-deficiency syndrome. The PI3K-Akt and MAPK signaling pathways and their related target genes may provide new insights into the molecular mechanisms of RAS with excess-heat syndrome or yin-deficiency syndrome, respectively.