RNA guanine (G)-quadruplexes (rG4s) are noncanonical structures formed by G-rich RNA sequences and have been demonstrated to play critical roles in various biological events, including translation, transcription, RNA processing, and other cellular functions. In contrast to DNA G-quadruplexes (dG4s), research on rG4s has been relatively limited until recently. Recent advances in targeting and imaging of rG4s have opened new avenues for understanding their functional significance and therapeutic potential. In this review, we summarize the innovative platforms and tools being developed to target rG4s, highlight the novel and important imaging probes that have been generated and applied for rG4 structure visualization in different biological contexts, and discuss the challenges and perspectives for further advancing these technologies and toolsets to facilitate rG4 targeting and imaging with greater precision and resolution across the Tree of Life. These scientific developments and breakthroughs will enable the discovery of new biological insights regarding rG4s and help decipher their molecular mechanisms and implications for health and disease.