The fluorescence quenching behavior of rhodamine 6G (R6G) by graphene oxide (GO) under varying pH conditions was investigated. Utilizing steady‐state fluorescence spectroscopy, single‐photon counting, and ultrafast time‐resolved absorption spectroscopy, we explored the quenching efficiency at pH values of 3, 7, and 11. Our findings reveal that GO effectively quenches R6G fluorescence across all tested pH levels, with the most significant quenching observed at pH 7. This quenching efficiency is attributed to optimal electrostatic interactions and efficient charge transfer between GO and R6G at neutral pH. At pH 3, the quenching efficiency is moderately reduced due to partial protonation of GO, which weakens electrostatic interactions but maintains hydrogen bonding. At pH 11, the quenching efficiency is lowest, likely due to increased electrostatic repulsion and reduced charge transfer resulting from deprotonation of GO. Ultrafast time‐resolved absorption spectroscopy further confirmed the dynamic nature of the quenching process, showing distinct differences in relaxation kinetics across the pH spectrum. This study highlights the critical role of pH in modulating the quenching mechanisms of GO and R6G, providing valuable insights for the design of pH‐sensitive fluorescence sensing systems.