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[Display omitted] • A new affinity peptide against β-LG was successfully screened, designed, synthesized, and characterized. • A conductive TA–PEI network was incorporated into electrode for electrochemical sensing of β-LG. • The developed sensor demonstrated a linear correlation range of 50 to 750 ng with a K d value of 213.9 ng. • The sensor could detect β-LG in cow and goat milk, with mean recoveries of 88.5% and 92.2%. Harmful substances that cause food allergies can pose a significant threat to consumers along with food safety. According to the World Health Organization (WHO), approximately 10 % of the global population is currently affected by food allergies. Therefore, there is an urgent need for the development of more accurate and precise biosensors capable of detecting these hazardous substances including beta-lactoglobulin. Although numerous detection and analysis methods have been developed, they still suffer from various limitations. In this study, a tannic acid–polyethyleneimine (TA-PEI) network modified screen-printed electrodes (SPE) are newly developed and the binding sequence of peptide against β-LG was successfully screened using random peptide library. A novel affinity peptide with the desired sequence of S-L-S-P-S-L-W-Q-V-S-M-L-G-G-G-G-E-P-L-Q-L-K-M against β-lactoglobulin (β-LG) is designed and synthesized. The synthesized affinity peptide was immobilized on TA-PEI modified SPE to develop peptide-based sensor against β-LG for the first time. Under successful optimization, the developed sensor exhibited a linear relationship between 50 and 750 ng, with a K d of 213.9 ng. In addition, the sensor was able to detect β-LG in cow and goat milk, with average recoveries of 88.5 % and 92.2 %, respectively. [ABSTRACT FROM AUTHOR]