The exposed hydrophobic patches of protein are widely detected through the binding by the fluorescent probes such as 1-anilino-8-naphthalene sulfonate (ANS), Nile Red (NR) and 1-(N-phenylamino) naphthalene, N-(1-Naphthyl) aniline (1NPN). Interestingly, at pH 4, where the Toxoplasma gondii Ferredoxin-NADP(+) reductase (TgFNR) is stable, an exclusive binding and fluorescence emission was observed for ANS. To understand the underlying difference in the binding of ANS, NR and 1NPN; their effect on the protein structure was studied in detail. ANS was found to interact with TgFNR via electrostatic as well as hydrophobic interactions at pH 4. NR and 1NPN did not show any such binding to TgFNR in the similar conditions, however showed strong hydrophobic interaction in the presence of NaCl or DSS (2, 2-dimethyl-2-silapentane-5-sulfonate). The subsequent structural studies suggest that ANS, NaCl and DSS induced partial unfolding of TgFNR by modulating ionic interactions of the enzyme, leading to the exposure of buried hydrophobic patches amicable for the binding by NR and 1NPN. The induced unfolding of TgFNR by ANS is unique and thus cautions to use the fluorescent dye as simple indicator to probe the exposed hydrophobic patches of the protein or its folding intermediates.