Dihydromyricetin, a flavonoid abundant in Ampelopsis grossedentata distributed across southern China, exhibits diverse pharmacological activities. In this study, a precise, sensitive, rapid, and reliable lateral flow immunoassay (LFIA) was developed for dihydromyricetin quantification in Ampelopsis grossedentata. A dihydromyricetin hapten was synthesized by introducing a carboxylic acid spacer via a four-carbon chain at the 3-position. A hybridoma cell line (2H3) secreting a highly specific monoclonal antibody (mAb) against dihydromyricetin was established. Dihydromyricetin demonstrated excellent stability in most organic solvents and acidic conditions. The LFIA exhibited a sensitivity (IC₅₀) of 33.61 ng/mL and a linear range of 4.54-281.83 ng/mL. The recovery rates of dihydromyricetin were in the range of 70.83-96.17 %. These findings indicate that LFIA, leveraging the high specificity of the dihydromyricetin mAb, offers a simple, rapid, and accurate method for dihydromyricetin analysis in Ampelopsis grossedentata.