To develop a novel high-throughput tool for monitoring specific affinity of microbes with lectins, a kind of lectin microarray has been fabricated by immobilizing lectins on epoxide-derivatized glass slides and used to capture microbes. The capturing events are marked by attachment of lectin-conjugated gold nanoparticles followed by silver deposition to enhance the resonance light scattering (RLS) of the particles. The interactions of 16 lectins with four bacteria and one fungus were profiled by this approach. We demonstrated that the gold-nanoparticle-labeled array was suitable for identifying the binding affinity of lectin with bacterium, as well as determining the bacterium with high sensitivity. More importantly, we found that the growth of microbial strains in different culture media resulted in significant changes in their binding affinities with lectins, which might be important to the pathogenesis of the organisms.