The invention relates to a preparation method of a Phlorigidoside C natural product standard substance in lamiophlomis rotata. The preparation method comprises the following steps: (1) sample treatment: cutting whole herb of the lamiophlomis rotata, and performing ethanol reflux extraction to obtain a lamiophlomis rotata extract; (2) solvent extraction: dissolving the lamiophlomis rotata extract and carrying out extraction and pressure reduced concentration to obtain a n-butanol extract; (3) macroporous resin column separation: dissolving the n-butanol extract, performing elution through a D101 macroporous resin column and performing pressure reduced concentration to obtain a Phlorigidoside C crude product; (4) reversal phase C18 column separation: dissolving the Phlorigidoside C crude product, performing elution through a C18 separation column, and performing pressure reduced concentration to obtain primarily purified Phlorigidoside C; and (5) purification through reversal phase preparative HPLC: dissolving the primarily purified Phlorigidoside C, performing purification through the reversal phase preparative HPLC, and performing pressure reduced concentration to obtain the Phlorigidoside C natural product standard substance. The method is simple in processes and is easy to operate and has high product yield and high quality. The Phlorigidoside C can be used for therapy of rheumatoid arthritis.