We developed a simple impedimetric biosensor for the detection of bacterial lipopolysaccharide (LPS) based on lectin-functionalized gold nanoparticle (AuNP)–graphene (G) composites. The effects of AuNPs on G were investigated by comparison with the sensing surface in the absence of AuNPs. The results showed that the AuNPs formed a 3D micro/ nanocomposite on G, which increased the electroactive area from 7.03 × 10−2 to 1.41 × 10−1 cm2, and also increased the heterogeneous electron transfer rate, which can improve the sensitivity of the biosensor. Concanavalin A (Con A) lectin was used as a molecular recognition element because of its specific interaction toward the carbohydrate groups in LPS. The increase in electron transfer resistance was in a logarithmically direct proportion to the LPS concentration and lowered the detection limit to 600 pg/L. The fabricated biosensor achieved the detection of LPS with good sensitivity, acceptable reproducibility, and stability. This method may hold promise for potential applications in glycomic biosensing.