A novel label-free fluorescence probe based on structure-switching aptamer was developed for the detection of interferon-gamma (IFN-γ). In this work, a single stranded DNA (ssDNA) with G-rich sequence (aptamer) was folded into secondary G-quadruplex structures in the presence of Na⁺ and Mg²⁺, thiazole orange (TO) was then intercalated into the G-quadruplex structures, resulting in a high fluorescence emission. The target combined with its aptamer, disrupts G-quadruplex structure and releases TO, resulting in a reduction of fluorescence. Using IFN-γ as the model target, the proposed fluorescence probe shows a linear range from 3.0 to 120 nM with a detection limit of 2.0 nM. The proposed strategy avoids complicated covalent modifications or chemical labeling, and thus offers advantages of simplicity and cost efficiency.