The interaction between tartrazine and bovine serum albumin (BSA) was investigated by fluorescence spectrometry, synchronous fluorescence spectrometry, three-dimensional fluorescence spectrometry, ultraviolet spectrometry and molecular docking. The analysis of fluorescence spectrum of tartrazine-BSA showed that tartrazine could effectively quench the endogenous fluorescence of BSA, and the fluorescence quenching constant K_SV decreased with the increase of temperature, so the quenching mechanism was static quenching on the basis of stern-volmer equation; According to the static quenching double logarithm formula, the binding constant (K_A) was calculated to be 4.335×10⁷ L·mol^-1 (293 K) and the number of binding point (n) was approximately equal to 1, which indicated that tartrazine had a strong binding ability with BSA and formed a binding site; The thermodynamic parameters obtained by van’t Hoff’s law (ΔH=-154.5 kJ·mol^-1, ΔS=-387.8 J·mol^-1·K^-1, ΔG<0) revealed that the main forces between tartrazine and BSA were hydrogen bond and van der Waals force, and the binding process was spontaneous; The binding distance (r) between tartrazine and BSA was calculated to be 3.310 nm based on the theory of Förster’s non-radiation energy transfer, which indicated that energy was likely to be transfered from BSA to tartrazine; With the increase of the concentration of tartrazine, the synchronous fluorescence intensity of Tyr and Trp residues decreased; The three-dimensional fluorescence spectra analysis showed that the intensities of peak 1 and peak 2 decreased significantly with the addition of tartrazine, and the emission wavelength of peak 2 changed, indicating that the peptide chain structure of BSA changed, and at the same time, the UV absorption peak of BSA increased gradually; The results of spectral analysis showed that the combination of tartrazine and BSA changed the conformation of BSA, thus changed the microenvironment around Trp and Tyr residues, resulting in the decrease of luminous efficiency; The results of molecular docking further illustrated that tartrazine was interacted with amino acid residues on subdomain Ⅲb of BSA and the amino acid residues around tartrazine mainly included: Phe506, Thr507, Ala527, Leu528, Met547, Gly571, Pro572, Leu574, Val575, Thr578; Tartrazine could interact with Thr507 and Thr578 residues by van der Waals force, with Thr507 by hydrogen bond and with other nonpolar amino acid residues by hydrophobic force. This research was helpful to understand the mechanism of interaction between tartrazine and BSA and reveal the distribution, metabolism and toxicological mechanism of tartrazine in vivo.