Many pathogens use mammalian cell surface carbohydrates as anchors for attachments, which subsequently results in infection. The unique combination of magnetic nanoparticles and diverse carbohydrate bioactivities prompts us to develop a magnetic glyconanoparticle (MGNP)-based system to rapidly detect Escherichia coli (E. coli ) in just 5 min. High capture efficiencies up to 88% were achieved. Moreover, to accurately differentiate three E. coli strains, response patterns of MGNPs were utilized to decipher the pathogen identity. This appealing approach may have clinical applications since the virulence of many pathogens can be correlated with carbohydrate binding specificity. To the best of our knowledge, this is the first time that MGNPs have been used to detect, quantify, and differentiate E. coli cells, which can provide an exciting new avenue for decontamination and diagnostic applications.