Abstract: Aloesone synthase (ALS), as a type Ⅲ polyketide synthase, plays an important role during the biosynthesis of diverse and active polyketide basic structural frameworks. In order to explore the expression pattern and function of RoALS2 in Rheum officinale, real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR), prokaryotic expression, and other techniques were used to analyze the expression pattern, in vitro protein expression, etc. of RoALS2 based on the previous transcriptome data. The overall results showed that the total length of the RoALS2 gene was 1 176 bp in size, encoding 391 amino acids, without signal peptides or transmembrane domains. Through evolutionary analysis and comparison, it was found that the RoALS2 is closely related to the ALS protein of Polygonum cuspidatum and R. palmatum. The RT-qPCR results showed that the relative expression level of RoALS2 in the roots and rhizomes of R. officinale increased first and then decreased with the change of growth years. The relative expression level was highest in the roots and rhizomes of two-year-old samples. The expression patterns of RoALS2 under different hormones' treatment were also different, and the overall trend was first upregulated, then downregulated, and then upregulated again. RoALS2 could respond to be upregulated by MeJA and SA treatments, but down-regulated by ABA and ethephon significantly. RoALS2 was then constructed into prokaryotic expression vectors pET-28a(+) and pGEX6p-1 respectively, and transformed into BL21 (DE3) competent, both of which showed successfully induction of the target fusion proteins. However, the pET-28a(+)-RoALS2 strain could be detected of the target band in the supernatant and the soluble protein was also obtained, whereas pGEX6P-1-RoALS2 recombinant protein existed in the form of inclusion bodies in the precipitate. The research results lay a solid foundation for further function characterization of the gene and elucidation of the molecular mechanisms of quality development of rhubarb from R. officinale.