Sulfonamide antibiotics (SAs) are widely used in medicine, animal husbandry and aquaculture, and excessive intake of SAs may pose potential toxicity to organisms. The toxicological mechanisms of two classical SAs, sulfamerazine (SMR) and sulfamethoxazole (SMT), were investigated by molecular docking, DFT and multi-spectroscopic techniques using HSA and BSA as model proteins. The quenching of HSA/BSA endogenous fluorescence by SMR was higher than that by SMT due to the stronger binding effect of the pyrimidine ring on HSA/BSA compared to the oxazole ring, and that result was consistent with that predicted by DFT calculations. Thermodynamic parameters show that the binding of SAs to HSA/BSA is an exothermic process that proceeds spontaneously (ΔG < 0). Marker competition experiments illustrate that the binding site of SMR/SMT on serum albumin is located in subdomain IIIA. The combination of SAs and HSA/BSA is mainly realized by hydrogen bond and hydrophobic interaction, and the concept is also supported by molecular modeling. The reduced α-helix content of HSA/BSA induced by SMR/SMT indicates a greater stretching of the protein α-helix structure of the SMR/SMT-HSA/BSA. The results could provide useful toxicological information on the hazards of SAs in response to growing concern that SAs may pose a toxic threat to organisms.