Abstract: In order to explore the protein differences of different processing methods at the protein level on peony petal(hot air drying, HD; natural drying, ZD; vacuum freeze drying, FD), and provide theoretical basis for subsequent processing, the proteomic differences of three kinds of processed peony petal were compared by label-free proteomics. The results showed that: A total of 1423 proteins were identified and 1349 proteins (94.80%)were quantified in the three groups. 1325, 677, and 1085 proteins were quantified in FD, HD and ZD respectively. There were 656 proteins in the three groups. Compared with HD, 77 differentially expressed proteins (DEPs) were detected in ZD, up-regulated 24 proteins, down-regulated 53 proteins. 77 DEPs annotation using GO and KEGG database revealed that proteins were enriched in glycolysis/gluconeogenesis pathway, pyruvate metabolism, TCA cycle, protein processing in endoplasmic reticulum. Conclusion: Processing methods had significant effect on protein difference of peony. This study is the first time to analyze the expression changes of differential proteins in peony flower by proteomics method, and provide scientific basis for different processing methods in practice.