G-quadruplexes (G4s) are widely found in cells and have significant biological functions, which makes them a target for screening antitumor and antiviral drugs. Most of the previous research on G4s has been conducted mainly in diluted solutions. However, cells are filled with organelles and many biomolecules, resulting in a constant state of a crowded molecular environment. The conformation and stability of some G4s were found to change significantly in the molecularly crowded environment, and interactions with ligands were disturbed to some extent. The structure of the G4s and their biological functions are correlated, and the effect of the molecularly crowded environment on G4 conformational transitions and interactions with ligands should be considered in drug design targeting G4s. This review discusses the changes in the conformation and stability of G4s in a physiological environment.