Let-7e precursor microRNA has the potential to adopt a G-quadruplex (rG4) structure and recently, its roles in oncology have been the focus of much attention, as it is now known that let-7e pre-miRNA is frequently dysregulated in cancers. Therefore, it is crucial to unveil and fully characterize its ability to adopt a rG4 structure, which could be stabilized or destabilized by small molecules and proteins such as nucleolin, a protein that is deeply associated with miRNA biogenesis. Herein, by combining a set of different methods such as circular dichroism (CD), nuclear magnetic resonance (NMR), UV spectroscopy (thermal difference spectra (TDS) and isothermal difference spectra (IDS)) and polyacrylamide gel electrophoresis (PAGE), we demonstrate the formation of the rG4 structure found in let-7e pre-miRNA sequence in the presence of K(+) (5'-GGGCUGAGGUAGGAGG-3'). The ability of eight small molecules (or ligands) to bind to and stabilize this rG4 structure was also fully assessed. The dissociation constants for each RNA G-quadruplex/ligand complex, determined by surface plasmon resonance (SPR), ranged in the 10(-6) to 10(-9) M range. Lastly, the binding of the rG4 structure to nucleolin in the presence and absence of ligands was evaluated via CD, SPR, PAGE and confocal microscopy. The small molecules 360 A and PDS demonstrated attractive properties to targetthe rG4 structure of let-7e pre-miRNA and control its biology. Our findings also highlighted that the interaction of TMPyP4 with the G-quadruplex of let-7e precursor miRNA could block the formation of the complex between the rG4 and nucleolin. Overall, this study introduces an approach to target the rG4 found in let-7e pre-miRNA which opens up a new opportunity to control the microRNA biogenesis.