Based on cell experiments, network pharmacology, bioinformatics analysis and molecular docking techniques were used to explore the bioactive components and potential mechanism of twigs of Broussonetia papyrifera (TBP) against anti-atopic dermatitis (AD). The anti-AD activity of TBP was validated using an interleukin-4 (IL-4)/tumor necrosis factor-alpha (TNF-α)-induced immortalized human keratinocytes (HaCaT cells) model. Databases including TCMSP, SwissTargetPrediction, UniProt, and STRING, combined with Cytoscape software, were employed to predict and screen core components and targets of TBP against AD. GO functional annotation and KEGG pathway analysis were performed using R language, followed by molecular docking validation between core components and targets. Machine learning and bioinformatics techniques were applied to identify key biomarkers and related pathways, while immune infiltration analysis was conducted via the CIBERSORT algorithm. Cellular experiments revealed that TBP's chloroform and ethyl acetate extracts significantly inhibited IL-13 secretion without cytotoxicity. Through network pharmacology analysis, key anti-AD constituents such as broussoflavan A and broussonin B were identified along with core targets, including serine/threonine kinase 1 (AKT1), proto-oncogene tyrosine-protein kinase SRC(SRC), and signal transducer and activator of transcription 1 (STAT1). GO analysis highlighted biological processes related to serine phosphorylation regulation, with membrane rafts and phosphatase binding as key cellular components and molecular functions. KEGG pathways were enriched in immune regulation and inflammatory response. Molecular docking confirmed strong binding affinity between broussoflavan A and core targets. Bioinformatics analysis identified STAT1 as a key biomarker, significantly associated with interferon-gamma response and E2F targets pathways, and positively correlated with activated CD4⁺ memory T-cell infiltration. These findings suggest that TBP and broussoflavan A may alleviate AD by targeting STAT1, SRC, and AKT1 to synergistically inhibit nucleotide-binding oligomerization domain-like receptors (NLRs) and Toll-like receptors (TLRs) signaling pathway, reduce pro-inflammatory cytokines (e.g., IL-13, IL-1β), regulate CD4⁺ T-cell and mast cell infiltration, and restore T helper 1/T helper 2 (Th1/Th2) immune balance and epidermal barrier integrity.