Potassium ion (K⁺) plays a central role in several fundamental physiological processes. Detection of the K⁺ concentration is an essential diagnostic tool for various medical diseases. However, most commercial detection methods are complex and expensive, which are not easily implemented in community hospitals or at home, in this study, we present a simple fluorescent K⁺ detection system based on the formation of G-quadruplex between K⁺ and dual-labelled thrombin aptamer oligonucleotide derivative (5′-FAM-TTTTTTAGGTTGGTGTGGTTGG-TAMRA-3′). Furthermore, based on this method, highly sensitive and selective detection of K⁺ in actual serum was realized by using EDTA as chelating agent to avoid the interference of Ca²⁺ and Mg²⁺ at physiological concentrations. Thus, this study paves the road toward the design and manufacture of portable potassium ions sensors based on fluorescence.