Drought stress was simulated with different concentrations (0, 5%, 10%, 15%, 20%, 25%) of PEG6000 solution. We used three cultivars of alfalfa (Medicago sativa) to clarify the effects of drought stress on the activities of three key enzymes as well as the accumulation of flavonoids. Results revealed that: (1) with the increase of PEG6000 simulated drought stress concentration, the activities of PAL (phenylalanine ammonia lyase), C4H (cinnamic acid4hydroxylase) and 4CL (4coumaric acid coenzyme A ligase) in the three alfalfa cultivar leaves increased first and then decreased but there were differences among different enzymes. PAL reached the maximum under 10% PEG stress, whereas, C4H and 4CL reached the maximum under 15% PEG stress and significantly higher than the corresponding control. (2) Compared with the control, with the increase of PEG6000 simulated drought stress concentration, the content of flavonoids in three cultivars of alfalfa increased first and then decreased, and the content of flavonoids was the highest under the stress of 10% to 15% PEG6000 and significantly higher than the corresponding control. (3) The activities of three key enzymes in the upstream synthesis of flavonoids in alfalfa leaves are closely related to the content of flavonoids and the content of flavonoids in the leaves of three alfalfa cultivars are related to their key enzyme activities to varying degrees. The results showed that different degrees of drought stress can increase the synthesis of flavonoids in alfalfa plants by promoting the accumulation of key enzyme activities upstream of flavonoids in alfalfa and moderate drought stress can significantly increase the related enzyme activities to promote the content of flavonoids.