Food allergy is an immune system reaction to a particular food, milk being the most common one. β-Lactoglobulin (β-Lg) is the main ingredient of milk protein and the main cause of infant milk allergy. On such an occasion, the determination of β-Lg is very important and the electrochemical sensors are a good alternative for this purpose since they are sensitive, selective and inexpensive. In this work, an electrochemical aptasensor was fabricated for the quantitative detection of β-Lg in hypoallergenic formula (HF) milk. A tri-functional hairpin (HP) was designed, which was composed of an aptamer sequence, a nicking site and a DNA sequence (T1). In the absence of β-Lg, the aptamer part hybridized with T1 to form a stable stem-loop structure. However, in the presence of β-Lg, the capture of the aptamer sequence towards β-Lg caused the reconstruction of HP and thus the nicking sites were exposed. Then, the nicking enzyme was activated and T1 could be released, which bound with the end of the hairpin 1-methylene blue (HP1-MB)/HP2-MB conjugation on the Au nanoparticle (AuNP) modified electrode surface. Thus, the insulating property of the electrode was enhanced and the current response of MB decreased, which built the quantitative basis for β-Lg detection. In this way, the proposed aptasensor exhibited a wide linear range of 0.01-100 ng mL-1 and a low detection limit of 5.7 pg mL-1. This aptasensor also displayed high selectivity, reproducibility and stability, and became a promising platform for β-Lg detection in real food samples.