Beta-lactoglobulin (β-Lg), a prominent milk protein, is a major contributor to milk allergies. The quantitative assessment of β-Lg is a valuable method for assessing the allergenic potential of dairy products. In this study, a specific aptamer, β-Lg-01, with an affinity constant (KD) of 28.6 nM for β-Lg was screened through seven rounds of magnetic bead SELEX (MB-SELEX). A novel bio-layer interferometry (BLI)-based aptasensor was developed, which had a limit of detection (LOD) of 0.3 ng mL-1, a linear range of 1.5 ng mL-1-15 μg mL-1, and a recovery rate of 102-116% among the milk samples. This aptasensor provides a potential tool for the detection and risk assessment of β-Lg within 10 min.