Abstract: Objective: To optimize the extraction process of total flavonoids from Pueraria lobata (TFPL), and evaluate its therapeutic potential in alleviating aluminum-induced intestinal barrier dysfunction. Methods: The extraction process was optimized via orthogonal design, with the yield of total flavonoids as the index. An in vitro intestinal barrier injury model was established using aluminum-exposed HT-29 cells. The protective effect of TFPL on the intestine was comprehensively assessed through a multi-parameter evaluation system, which encompassed in vitro antioxidant capacity assays, intestinal epithelial monolayer permeability measurements, quantitative analysis of intracellular reactive oxygen species (ROS) levels, and molecular profiling of both inflammatory cytokines and tight junction protein expression. Results: The optimized extraction parameters were established as follows: Ultrasonic power of 400 W, solid-to-liquid ratio of 1:30 g/mL, extraction duration of 60 min, and ethanol concentration of 60%. Under these optimal conditions, the maximum yield of total flavonoids reached 4.65±0.05 mg/g. The in vitro antioxidant assays indicated TFPL was capable of scavenging DPPH, ABTS⁺, and hydroxyl radicals. In aluminum-exposed HT-29 cells, TFPL was demonstrated to significantly (P<0.05) improve reduced TEER value, increased FITC-dextran permeability, elevated ROS levels, upregulated secretion of TNF-α and IL-1β, activated NF-κB pathway, and downregulated expression of claudin-4 and occludin. Conclusion: TFPL exhibited protective effects on the intestine against aluminum-induced damage. The underlying mechanism might be associated with oxidative stress mitigation, anti-inflammatory actions, and tight junction modulation.