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Tree peony (<i>Paoenia ostii</i>) is a famous Chinese traditional flower well-known in many countries of the world. However, the short and concentrated flowering period of tree peony greatly affects the ornamental and economic value of the flowers. Exogenous brassinosteroid (BR) treatment can delay the flowering period of <i>ostii</i> T. Hong et J. X. Zhang var. <i>lishizhenenii</i> B. A. Shen for 3 days, but the underlying regulatory mechanism remains elusive. Here, full-length transcriptome and transcriptome sequencing were used to mine key genes related to BR-induced delayed flowering in tree peony. The transcriptome sequencing of the petals yielded 21.27 G clean data and 62,229 isoforms. Among them, 58,218 isoforms were annotated in NR, NT, SwissProt, KEGG, KOG, InterPro and GO databases. GO and KEGG analyses showed that 2460 DEGs were related to delayed flowering in response to BR. Additionally, a total of seven genes affecting flowering were annotated from 11 isoforms, which responded to BR through three pathways to delay the flowering of <i>P. ostii</i> var. <i>lishizhenii</i>. BR treatment increased the expression of <i>BRASSINOSTEROID-SIGNALING KINASE3</i> (<i>BSK3</i>), potentially by promoting <i>BRI1 ASSOCIATED KINASE RECEPTOR 1</i> (<i>BAK1</i>). Moreover, BR treatment suppressed the expression of <i>SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE 1 (SPL1)</i>, possibly to inhibit the expressions of downstream <i>WRKY</i> genes and <i>APETALA 2</i> (<i>AP2</i>). Meanwhile, BR treatment promoted the expression of <i>DELLA</i>, which might inhibit the expressions of <i>CONSTANS</i> (<i>CO</i>) and <i>SPL</i>. These results suggest a theoretical basis for further analyses of the molecular mechanism of flowering regulation in tree peony.