Abstract: Medicago polymorpha L. from Shanghai was used as the experimental material, and PEG-6000 was used to simulate drought stress. Based on the PEG-6000 threshold, the changes in seed germination, plantlet growth indexes, physiological and biochemical indexes, and gene expression of Medicago polymorpha L. were studied after adding concentrations of 0%, 0.1%, 0.2%, 0.4%, 0.8% and 1.6% GABA to MS solid medium. The results showed that under 5% PEG-6000 drought stress, the CAT activity, SOD activity, POD activity, MDA content, soluble sugar content, soluble protein content, and proline content as well as the expression of WRKY transcription factor, protein TIFY 8, transcription factor MYB13, NAC transcription factor 47, ABC transporter A family member 7, late embryogenesis enrichment protein 2, and ethylene responsive transcription factor ERF110 of seed aseptic germination and plantlets of Medicago polymorpha L. significantly increased. Under 5% PEG- 6000 simulated drought stress, 0.2% GABA and 0.4% GABA were beneficial for increasing CAT activity, SOD activity, POD activity, soluble sugar content, soluble protein content, proline content, and the expression of WRKY transcription factor, protein TIFY 8, transcription factor MYB13, NAC transcription factor 47, ABC transporter A family member 7, late embryogenesis rich protein 2 and ethylene responsive transcription factor 110 in seed aseptic germination plantlets of Medicago polymorpha L. and its plantlets, while reducing their MDA content. Under simulated drought conditions with 5% PEG-6000 and the addition of different concentrations of GABA, the growth indexes were extremely significantly positively correlated with antioxidant enzyme activity, osmoregulatory substances and gene expression in seed germinating plantlets of Medicago polymorpha L. and its plantlets, and were significantly negatively correlated with MDA content. In conclusion, external application of 0.2% GABA and 0.4% GABA can improve the drought resistance of seeds and plantlets of Medicago polymorpha L. under drought stress, which could be applied in practical production to enhance the drought resistance of Medicago polymorpha L.