Sunset Yellow (SY) is a potentially harmful artificial synthetic pigment. This paper, taking human serum albumin (HSA) as the research object, analyzes the quenching effect of SY on HSA and determines the quenching constant Ksv of the quenching reaction with the fluorescence spectroscopy method. The results show that the quenching effect of SY on HSA is static quenching. In addition, based on the analysis of the binding of SY and HSA with the synchronous fluorescence spectroscopy method, it is found that the binding of SY and HSA is near tryptophan, whose residue is caused to be in a stretching state and the structure of HSA has changed. Furthermore, this paper selects ten types of metal ion, adds them into the reaction system of SY and HSA under the simulated human physiological conditions, and detects the impacts of the metal ions on the system with the three-dimensional fluorescent spectrometry method, which demonstrating that Cu2+, Pb2+, Ni2+ and Mn2+ exert facilitation effects on the quenching process, and Ni2+ shows the largest impacts, with an increase of 22.6%, while Fe2+ and Zn2+ have inhibition effects on the quenching process, with an inhibition ratio of 14.12% and 14.2%, respectively. Therefore, the experimental method is applicable to the study on the toxicity of food additives and conducive to ensureing food safety as well as maintaining human health.