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In the present study, a food additive, tert-butylhydroquinone (TBHQ), was selectively and sensitively determined by both colorimetric and spectrofluorimetric methods using bovine serum albumin capped gold nanoclusters (BSA-AuNCs) for the first time. The AuNCs were synthesized by a one-pot method using BSA as a stabilizing agent in an alkaline medium at room temperature. The synthesized BSA-AuNCs were then confirmed by UV-vis spectroscopy, fluorimetry and HR-TEM. The absence of a surface plasmon resonance band and appearance of red luminescence in a UV chamber confirmed the formation of AuNCs. The HR-TEM images exhibited the average size of BSA-AuNCs as 1.8 nm. The BSA-AuNCs showed an emission maximum at 663 nm and were utilized for the colorimetric and fluorimetric detection of TBHQ. The addition of 50 μM TBHQ to the BSA-AuNCs leads to the disappearance of the red luminescence as well as the quenching of the emission at 663 nm. This is due to the strong electrostatic interaction between TBHQ and BSA-AuNCs and it was confirmed from the decrease in the zeta potential value from −43.7 to −0.9 mV. The emission intensity of the BSA-AuNCs does not change when adding higher concentrations of major interferents including propyl gallate, butyl hydroxyltoluene, butyl hydroxyanisole and metal ions. On the other hand, the emission intensity decreases when increasing the concentration of TBHQ from 2.5 to 50 μM and the limit of detection was found to be 72 nM (S/N = 3). The practical application of the BSA-AuNCs was determined in oil samples for determining TBHQ and the results were validated with the GC-MS method.