A dual-luciferase reporter gene system to rapidly screen for probiotics with anti-inflammatory activity was constructed in this study. The conditions for the transfection of dual-luciferase plasmids containing nuclear factor-kappa B (NF-κB) response elements into 293T cells and the concentration of lipopolysaccharide (LPS) as an inducer were optimized. The anti-inflammatory activity of 86 selected strains was comparatively evaluated using this system and macrophage RAW264.7 cells. The results showed that the optimal transfection conditions were determined as 50:1, 1:1, 24 h and 100 ng/mL for pNF-κB-luc to pRL-TK plasmid ratio, plasmid to transfection reagent concentration ratio, transfection time, and LPS concentration, respectively. The dual-luciferase reporter system was reliable (Z’= 0.6632) and stable (R² = 0.74699), and its results were consisted with those obtained using macrophage RAW264.7 cells. Finally, three strains with excellent anti-inflammatory effect, Lactiplantibacillus plantarum X30, Limosilactobacillus fermentum X58 and Weissella confusa X83, were obtained by this method. Each strain effectively inhibited the activation of the NF-κB pathway, significantly reduced the expression of pro-inflammatory factors such as interleukin (IL)-1β, tumor necrosis factor-α, and NF-κB p65, and increased the expression of IL-10. This system provides a new idea for targeted screening for probiotics with anti-inflammatory activity.