Abstract: Zinc （Zn） and copper （Cu） have been extensively used as feed additives. Owing to their low utilization efficiency by animals, substantial quantities of Zn and Cu were excreted in feces and subsequently entered the soil, leading to Zn and Cu pollution. This study used alfalfa （Medicago sativa L.） seeds as experimental material to examine the effects of combined Zn and Cu stress on various growth and physiological parameters of alfalfa seedlings, as well as their tolerance to Zn and Cu stress. Furthermore, correlation analysis and principal component analysis were conducted to provide a foundation for future research on the potential of alfalfa for remediating soil contaminated with Zn and Cu. The experiment was conducted using a hydroponic system, in which alfalfa seedlings were exposed to varying concentrations of Zn²+（0, 100, 200, 500 mg/L） and Cu²+（0, 50, 100, 200 mg/L） in a modified Hoagland solution. The findings indicated that the alfalfa seedlings accumulated Zn and Cu to some extent, with Zn accumulation being more pronounced under combined stress conditions. Correlation analysis demonstrated that, under the combined Zn and Cu stress, the concentrations of heavy metals, hydrogen peroxide （H₂O₂）, and malondialdehyde （MDA） in the seedlings were positively correlated with the stress concentration. Conversely, the stress concentration was negatively correlated with both relative root cell viability and chlorophyll content. Principal component analysis identified hydrogen peroxide （H₂O₂）, proline, and catalase （CAT） as pote

Key words: heavy metal stress, plant tolerance, physiological responses, correlation analysis, principal component analysis