In recent years, the overuse of antibiotics in livestock feed has raised significant public health concerns. As a result, antibiotic-free breeding has become a key focus of sustainable agricultural development. Efforts to replace antibiotics in feed have largely focused on plant-derived compounds, extracts, and probiotics or their metabolites. In this study, four probiotic strains: Lactobacillus delbrueckii XH-9, Lacticaseibacillus plantarum GM-6, Lactiplantibacillus rhamnosus GM-7, and Bacillus subtilis N-1 were isolated from traditional fermented products of the Tibetan Plateau, and demonstrated inhibitory effects against Escherichia coli and Salmonella. A total of 45 healthy male Hu sheep (aged 30 days, with similar initial body weight) were randomly assigned to three groups: control, antibiotic, and probiotics. After 35 days of feeding, the probiotic group showed significantly reduced levels of high-density lipoprotein cholesterol (HDL-C; P < 0.001), total cholesterol (T-CH; P < 0.0001), total bilirubin (T-BiL; P < 0.001), creatinine (CR; P < 0.001), interleukin-1β (IL-1β; P < 0.05), and malonaldehyde (MDA; P < 0.001), alongside increased levels of total protein (TP; P < 0.05) and tumornecrosis factor-α (TNF-α; P < 0.0001), as well as enhanced activities of superoxide dismutase (SOD; P < 0.05), and catalase (CAT; P < 0.05). Analysis of gut microbiota composition demonstrated that antibiotic treatment significantly altered microbial community structure and promoted the growth of opportunistic pathogens. Conversely, probiotics markedly increased the relative abundance of beneficial bacteria, including Lactobacillus (P < 0.05) and Bifidobacterium (P < 0.0001). These findings collectively demonstrate that the probiotic strains effectively reduced oxidative stress, enhanced immune responses, and supported healthy gut microbiota development in growing lambs, indicating their potential as effective probiotic feed additives.