RNA G-quadruplexes (rG4s) are emerging structural motifs that are of pivotal importance in chemistry and biology; however, the current structural information of rG4s is limited, with their folding status and functions in cells remaining elusive. Here, we develop and employ a multi-disciplinary approach to characterize the structure, formation and function of an individual rG4 of interest in vitro and in cells. We apply this strategy to a biologically important rG4 in amyloid precursor protein (APP) transcript and reveal distinct structural features of APP rG4. Notably, we visualize the formation of APP rG4 in cells using an APP-specific G-quadruplex-triggered fluorogenic hybridization (GTFH) probe and report that the regulatory role of APP rG4 in translation is dependent on rG4 thermostability, providing evidence to the existence and significance of the stable rG4 structure in gene regulation.