Herbaceous peony (Paeonia lactiflora Pall.) is popular worldwide because of its gorgeous flower colour, and the yellow flower is the rarest. However, its mechanism of yellow formation is still unexplored from the post-translational level. In this study, the anatomy of the petal, cell sap pH and metal elements were investigated in bicoloured flower cultivar 'Jinhui' with red outer-petal and yellow inner-petal, and the yellow formation was influenced by the anatomy of petal, while not by the cell sap pH and metal elements. Subsequently, microRNAs sequencing (miRNA-seq) was used to identify small RNAs (sRNAs). A total of 4,172,810 and 3,565,152 specific unique sRNAs were obtained, 207 and 204 conserved miRNAs and 38 and 42 novel miRNAs were identified from red outer-petal and yellow inner-petal, respectively, which were confirmed by subcloning. Among these miRNAs, 163 conserved and 28 novel miRNAs were differentially expressed in two wheel of petals. And 5 differentially expressed miRNAs and their corresponding target genes related to yellow formation were screened, and their dynamic expression patterns confirmed that the yellow formation might be under the regulation of miR156e-3p-targeted squamosa promoter binding protein-like gene (SPL1). These results improve the understanding of miRNA regulation of the yellow formation in P. lactiflora.