This study screened small molecule compounds capable of specifically binding to and stabilizing the G-quadruplex (G4) structure within the Rv0341 gene of Mycobacterium tuberculosis. Firstly, circular dichroism (CD) spectroscopy was used to confirm the formation of the G4 structure (designated Rv0341G4) by the G-rich oligonucleotide sequence in Rv0341 gene. Subsequently, real time fluorescent quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were employed to validate the inhibitory effects of candidate small molecules on Rv0341 expression. Through surface plasmon resonance (SPR) screening, a small molecule compound T-007 exhibiting high-affinity binding to Rv0341G4 was identified. Both qRT-PCR and Western blot analyses demonstrated that T-007 stabilizes the Rv0341G4 structure and downregulates Rv0341 transcription. This study provides valuable insights for developing novel anti-tuberculosis therapeutics targeting G4 structures.