Herein, a novel label-free electrochemical immunosensor was fabricated via immobilizing specific anti-β-lactoglobulin (β-LG) antibodies (Abs) onto an integrated electrode of gold nanoparticles (AuNPs)/Prussian blue (PB)/cubic Ia3d structured mesoporous carbon (CMK-8). This immunosensor allowed for the quantitative detection of the major milk allergen β-LG. CMK-8 with excellent electrical conductivity and uniformly adjustable pore structure was modified on the glassy carbon electrode (GCE) and served as the sensitive substrate for the electro-polymerization of PB, forming the redox-active layer. AuNPs were subsequently electrochemically deposited on PB/CMK-8/GCE to improve the electrical conductivity and utilized as the connector for Abs immobilization. During β-LG detection, the Abs-modified AuNPs/PB/CMK-8/GCE exhibited a significant reduction in differential pulse voltammetry current signal when exposed to β-LG, displaying an inverse dose-dependent relationship. The developed electrochemical immunosensor demonstrated good detection performance for β-LG, with a wider linear range of 0.01–100 ng/mL and a lower detection limit of 4.72 pg/mL. Meanwhile, the sensor exhibited remarkable repeatability, reproducibility, stability and anti-interference capabilities, which was further applied to detect β-LG in dairy food, achieving satisfactory recoveries (89.2%–98.8%) and lower relative standard deviation (￡ 3.1%). Therefore, this innovative electrochemical method for food allergen detection holds great potential application in food safety determination and evaluation.