Metal contaminants present persistent and deleterious threats to environmental ecosystems and human health. Microorganisms can rapidly develop protective mechanisms against metal toxicity, such as metallothionein production. The identification of biological factors related to these protective mechanisms is essential for effective metal remediation. This study presents a robust pathway to rapidly locate and characterize a Pb-binding flagellin in Serratia Se1998, which can bind Pb at a 16:1 Pb: protein ratio. A column gel electrophoresis system hyphenated with inductively coupled plasma mass spectrometry (ICP MS) was constructed to efficiently separate and identify Pb-binding proteins from the whole bacterial proteome. PCR and transgenic assays were used to elucidate the exact sequences and biological function of Pb-binding proteins and heterogeneous expression of Pb-binding flagellin in E. colt could significantly enhance Pb removal from aqueous solution by approximately 45%. This method provides a benchmark procedure to rapidly identify biological factors responsible for metal bio-sorption. Identification of this unique Pb-binding flagellin highlights that microorganisms can survive high metal stresses due to various complex biological pathways for metal detoxification and remediation.