G-quadruplexes (G4s) are non-canonical nucleic acid secondary structures that play a crucial role in regulating essential cellular processes such as replication, transcription, and translation. The formation of G4s is dynamically controlled by the physiological state of the cell. Accurate detection of G4 structures in live cells, as well as studies of their dynamic changes and the kinetics of specific G4s, are essential for understanding their biological roles, exploring potential links between aberrant G4 expression and disease, and developing G4-targeted diagnostic and therapeutic strategies. This perspective briefly overviews G4 formation mechanisms and their known biological functions. We then summarize the leading techniques and methodologies available for G4 detection, discussing the principles and applications of each approach. In addition, we outline strategies for the global detection of intracellular G4s, methods for conformational recognition, and approaches for targeting specific sequences. Finally, we discuss the technical limitations and challenges currently facing the field of G4 detection and offer perspectives on potential future directions. We hope this review will inspire further research into the biological functions of G4s and their applications in disease diagnosis and therapy.