To verify the response of different concentrations of 5aminolevulinic acid (ALA), we sprayed alfalfa (cv. Nongmu 806) seedlings with 15% PEG6000 and ALA with different concentrations (0, 5, 10, 15, 20, 25 mg·L^-1), and 1/2 Hogland nutrient solution was used as control. Then, the physiological, biochemical indexes and secondary metabolite of ‘Nongmu 806’ seedlings were determined. Results revealed that: (1) compared with the control, 15% PEG simulated drought stress reduced the contents of chlorophyll a, b, carotenoid and chlorophyll in alfalfa leaves, increased the content of osmotic adjustment substances and the activities of antioxidant enzymes. (2) After ALA and 15% PEG treatment, the contents of chlorophyll a, b, carotenoid and chlorophyll of alfalfa seedling leaves increased, the activities of CAT, POD, SOD and the contents of soluble protein, proline, flavonoids and saponins increased, whereas the contents of malondialdehyde, H₂O₂ and·OH decreased compared with 15% PEG treatment. The results showed that spraying ALA can effectively improve the tolerance of alfalfa to drought stress. However, different concentrations of ALA have different recovery effects on 15% PEG, and 10 mg·L^-1 ALA had the most significant effect.