Pb(II) sequestration in extracellular polymers-biochar composites (EPS-BC) was explored using macroscopic models and microscopic technology. The results showed that the actual adsorption capacity of EPS-BC was 52.2% lower than the calculated capacity based on adsorption onto pure components due to the interaction of polysaccharide and amide group in extracellular polymers with biochar, which masked the reactive sites related to Pb(II) in EPS-BC. The bond of Pb–O (40.8%) and Pb–OOC (31.5%) mainly contributed to Pb(II) speciation on the EPS-BC surfaces. Furthermore, each Pb atom coordinated with 6O atoms in the first shell and with 0.5C atoms in the second shell, indicating that the carboxyl group in composite was complexed with Pb(II) as a monodentate inner-sphere structure. The findings provide an in-depth understanding of the adsorption mechanism of heavy metals by extracellular polymers coupled with biochar at molecular scale, guiding bioremediation with respect to heavy metal contamination.