Flavonoids are secondary metabolites that accumulate in most plants. Calycosin-7-O-β-D-glycoside (CG), as a flavonoid, plays an important role in the abiotic stress response of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus). CG is also an active ingredient in A. mongholicus with high medicinal value. However, the response mechanism of CG biosynthetic pathway to drought stress is not clear. In this research, drought stress was inflicted upon annual potted A. mongholicus for 15 days and the variations in flavonoid metabolites and the correlating gene expression in CG biosynthesis were studied in roots, stems and leaves of A. mongholicus by UHPLC-MRM-MS/MS and qRT-PCR. Drought stress reduced the dry weight and increased the content of MDA and Proline. Drought promoted the accumulation of most compounds in the CG synthetic pathway of A. mongholicus, which decreased after rewatering. AmI3’H is highly expressed in the roots under drought stress. Overexpression of AmIOMT was observed in the leaves, but the content of formononetin which is the product of IOMT catalysis was higher in stems than in leaves. This research aims to further understand the acclimation of plant to abiotic stress and the regulation mechanism of flavonoid accumulation in astragalus species.