Objective To explore the pathogenesis and syndrome differentiation of Pi-deficiency syndrome (PDS) from microRNA (miRNA) levels through screening and bioinformatic analysis of serum miRNA expression in PDS patients. Methods Four hyperlipemia patients with PDS, 4 hyperlipemia patients with Pi-Wei damp-heat syndrome (PWDS) and 5 healthy volunteers were recruited. Their serum RNA was used in miRNA quantitative PCR array experiment. Serum miRNA expression profiles in PDS patients were screened to perform bioinformatic analysis. Results Nine candidate miRNAs (6 upregulated and 3 downregulated) were screened from PDS patients. These miRNAs were able to clearly distinguish among PDS patients, PWDS patients and healthy volunteers. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed 83 target genes controlled by 6 up-regulated miRNAs were significantly enriched in 7 pathways, which were mainly involved in cytokine-cytokine receptor interaction, pathogens of infectious diseases, immune/inflammatory-related signaling pathway and pancreatic cancer; and 365 target genes controlled by 3 down-regulated miRNAs were significantly enriched in 5 pathways, which were mainly involved in signaling pathways of neurotrophin and phosphatidylinositol, RNA transport, and metabolisms of inositol phosphate and amino acid. Conclusion Our findings provide potential miRNA biomarkers for clinical syndrome differentiation of PDS patients, as well as information for understanding and studying the pathogenesis of PDS patients.