High expression of Interluekin-1 receptor accessory protein chain (IL-1RAcP) and activated IL-1 signaling were found in some tumor types. IL-1RAcP is considered as the common accessory chain in the IL-1R family, and it is essential for the initiation of IL-1 signaling of all the receptor complexes that encompass it. Thus, the selection and characterization of human anti- IL-1RAcP single-chain antibody fragments variable (scFv) is the first step toward the construction of new anticancer monoclonal antibodies designed for optimal cancer therapy. Here, we found that IL-1RAcP expression was increased in both triple-negative breast cancer (TNBC) cell line cells and TNBC patient cohort, and correlated with shorter recurrence-free survival (RFS). In this study, we employed a human scFv–displaying phage library for the first establishment an antagonistic anti–IL-1RAcP human antibody, scFv 12H7. scFv 12H7 was found a high affinity and specificity binder of IL-1RAcP by a series assays, including EC₅₀, IC_50, K_D values test and cell binding determination by flow cytometry and immunofluorescence. Also, scFv 12H7 was demonstrated bearing growth inhibitory activity of TNBC cells in vitro and in vivo. Mechanisms study showed that IL-1-activated-NF-κB pathway was significantly inhibited in TNBC cells by incubation with scFv 7H12 for 24 h. Crystal structure analysis, mutations introduction, and yeast two-hybrid assay showed that scFv 12H7 interacted with residues in the D1-D2 domain of IL-1RAcP, which further indicated that scFv 12H7 was a functional binding to IL-1RAcP and uncovered its structure mechanism. In conclusion, scFv 12H7 represent excellent therapeutic candidates for further preclinical and clinical development of TNBC therapy.