One of the pathogenesis hypotheses of Alzheimer's disease (AD) is amyloid depositions and neurofibrillary tangles. Apolipoprotein E (Apo E) acts a vital part in the development of AD by affecting the aggregation and clearance of amyloid-β (Aβ). In this paper, a dual polarization interferometry (DPI) technique was employed for a real-time investigation toward the binding events of Apo E isoforms, for instance, Apo E2, Apo E3, and Apo E4, with Aβ1-40. By evaluation of detailed binding information provided by DPI, the affinities between Apo E isoforms and Aβ1-40 follow the order of E4 > E3 > E2, and the dissociation constants (KD) of Aβ1-40 with Apo E2, Apo E3, and Apo E4 were determined to be 251 ± 37, 40 ± 0.65, and 24.6 ± 2.42 nM, respectively. Our findings reveal the isoform-specific binding behaviors from a kinetics perspective, which can help us understand that Apo E4 has a higher risk of causing AD because of its promoting effect on Aβ aggregation and fibrillation and inefficient clearance of Aβ. Remarkably, this work provides a promising method for exploring the dynamics of interactions between biomolecules and expectantly contributes to the development of AD drugs and therapies targeting Apo E and Aβ.