During the process of germination, seeds use up their protein reserves and experience various physiological and biochemical changes, leading to stress. This study examined the impact of Abscisic acid (ABA) and nanoparticle treatments on the regeneration of proteins consumed during the germination of alfalfa seedlings, as well as the activities of antioxidant enzymes in germinating seeds. Specific antioxidant enzymes play crucial roles in plant growth and development at different stages of growth and conditions. Alfalfa seeds were germinated and treated with Titanium dioxide (TiO2) and activated charcoal after 48 hours of germination. The protein was extracted from seedlings that were 5, 7, and 10 days old. The analysis showed that the seedlings treated with ABA and TiO2 had a higher protein content than other treatments but lower than dry seed, which serves as a control. An SDS-PAGE examination revealed the reappearance of some protein bands in seedlings treated with ABA and TiO2 after 5, 7, and 10 days of germination. The activities of catalase and peroxidase showed an increase as the seedlings grew older and were shown to be higher in seedlings treated with ABA and TiO2. The findings of our study indicate that the application of ABA and TiO2 alters the expression of proteins and increases the activity of antioxidant enzymes in young alfalfa seedlings, perhaps leading to an enhanced ability to withstand stress. These discoveries have implications for the growth of young plants and the enhancement of agricultural yields.