The steaming process is one of traditional Chinese medicine's processing approaches for many herbs. A new HPLC peak was found in the root of Polygonum multiflorum Thunb. (PM) after steaming. 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one (DDMP) and 5-hydroxymethyl furfural (5-HMF) were confirmed in the new peak. Additionally, two other compounds, butanedioic acid and 3,5-dihydroxy-2-methyl- 4(H)-pyran-4-one (hydroxymaltol), were isolated in the processed root of PM. An increase in DDMP concentration was observed during the first 24 h of steaming, followed by a constant decrease until 64 h of steaming. The amount of 5-HMF increased gradually throughout the steaming process. Changes to 16 kinds of amino acid were found after the steaming process. Drastic changes in the concentration of D-glucose, D-fructose, and sucrose were observed during the first 16 h, followed by steady value changes to 64 h of steaming using HPLC-ELSD. The pH values decreased from 6.28 to 5.61 during the 64-h steaming process. The production of DDMP, 5-HMF, butanedioic acid, and hydroxymaltol and the changes in amino acid, sugar, and pH values suggest that the Maillard reaction was involved in the steaming process of the root of PM, which may contribute to the activity differences between the root of PM and the processed root.