Objective To study the effect of Huanglian Jiedu Decoction(HLJD) on the physiological function of healthy SD rats through serum non-targeted metabolomics. Methods After 7 days of adaptive feeding, 24 rats were randomly divided into blank group (group N) and high-dose HLJD group (group H), medium-dose HLJD group (group M), and low-dose HLJD group (group L) with six rats per group. Group N was gavaged with physiological saline, and H, M, and L groups were gavaged with 6.250, 3.125, and 1.560 g/kg HLJD, respectively. Rats were gavaged with 1 mL twice per day for 21 d. Before collecting serum samples, fasting and water deprivation were performed for 12 h. After anesthesia, blood was collected from the abdominal aorta, and serum was separated. Changes in metabolic products in the serum were detected by Gas Chromatography Time-of-Flight Mass Spectrometry(GC-TOF-MS). Multivariate analysis by Orthogonal Projections to Latent Structures Discriminant Analysis(OPLS-DA) and univariate statistical methods were used to screen differential metabolites, and then metabolic pathway analysis was conducted using the Metabo Analyst platform. Results Compared with the group N, metabolic small molecules were significantly altered in the serum of group H, group M, and group L, which mainly included cholesterol, cholic acid, ribose, paclitaxel, cortisol, oleic acid, succinic acid, linolenic acid, acetic acid, and acetone alcohol (P < 0.05). Correlation analysis showed that HLJD significantly affected the metabolism of primary bile acids, alanine, aspartic acid, and glutamate in α-linolenic acid and glycerol phospholipid metabolism, and its in vivo effects may be related to the intake dose, providing a reference to further reveal the mechanism of HLJD in promoting bile acid metabolism. Conclusions HLJD promotes the production of primary bile acids in vivo and enhances the effects of the bile acid metabolic pathway.