Fluorescence anisotropy, dictated by the rotational diffusion of the fluorophore associated entity, is sensitive to the volume and structural changes, and the measurements can be performed in a homogeneous manner. In this work, a simple approach based on fluorescence anisotropy was proposed for the study of conformational switching of the i-motif structure. Factors influencing the stability of the i-motif structure, including the composition of buffer, the number of i-motif tetrads and coexistence of the complementary DNA, were investigated in detail. This study elucidated the superiority of the fluorescence anisotropy measurement as a simple, cost-effective and sensitive method for the detection of DNA structural switching, opening a new avenue for the research of DNA structures and functions.