PFOS and PFOA are persistent in nearly all water bodies and are very thermally and chemically stable, making them non-biodegradable and posing health risks. When bound with proteins, this relationship can affect human health. Several detection methods involving gas chromatography, mass spectrometry, and other PFOS detection procedures are time-consuming and require expensive instrumentation. Consequently, fluorescence spectroscopy was used to quantify PFOS by utilizing an SQ-BSA complex based on the sulfonated-squaraine dye (SQ) absorption and fluorescence properties. The SQ dye (blue-solid) was prepared by reacting with squaric acid and the synthesis of 3-(2-Methylbenzo[D]Thiazol-3-ium-3-yl)-propane-1-sulfonate in a Dean-Stark apparatus containing 5 mL of anhydrous pyridine, 4 mL of n-butanol, and 4 mL of toluene. The system was refluxed at 175°C for 5 hours, after which the reaction mixture was purified via recrystallization in methanol twice and then finally characterized by ¹H NMR. It interacted with bovine serum albumin (BSA) to form the SQ-BSA complex, which was then investigated separately and showed fluorescence turn-on. The complex emission continuously increases as the BSA concentration rises, accompanied by a corresponding wavelength shift from 640 nm to 660 nm due to a non-covalent interaction. However, subsequent PFOS and PFOA additions revealed a gradual fluorescence turn-on and turn-off, respectively, which continued to increase along these two directions up to a 20 µM concentration. Overall, the detection of these sequentially studied tri-component systems (SQ, BSA, PFOS/PFOA) relies on the different interacting behaviors of SQ with PFOS and PFOA, thereby providing a complementary approach for detecting PFOS and PFOA in water. Therefore, the demonstrated very strong discrimination ability of PFOS and PFOA by the SQ-BSA complex becomes ascribed to the obviously known differing interacting abilities of these two harmful pollutants with BSA amongst various studies.