Update publication information. Toad venom is a traditional Chinese medicine with high medicinal value. The existing quality evaluation standards of it has obvious limitation for lack of protein study. Thus, it’s necessary to screen suitable biomarkers and establish appropriate quality evaluation method for the characteristics of toad venom’s proteins, guaranteeing the safety and efficacy in clinical applications. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-performance liquid chromatography and cytotoxicity assay were used to analyze protein components difference between toad venom of different areas. Functional proteins were screened as potential biomarkers by tandem mass tags proteomic and bioinformatic analysis. The results showed that the composition of different toad venom was similar. The protein content of Lianyungang’s toad venom was higher and the alcohol-soluble substance was lower, which suggested that they had no correlation. Simultaneously, the protein component was verified to have strong cytotoxicity. Proteomics analysis showed that 13 antimicrobial proteins, 4 anti-inflammatory and analgesic proteins, and 20 antitumor proteins were screened in the extracellular differentially expressed proteins. A candidate list of functional proteins that could be used as potential biomarkers was presented. Moreover, the antimicrobial activity of Lysozyme C-1 and the anti-inflammatory and analgesic activity of Neuropeptide B were identified as biomarkers for toad venom protein in future. The biomarkers can form the basis for the quality studies of toad venom’s proteins, helping construct and improve the safe, scientific and comprehensive quality evaluation method.