Carbohydrate microarrays have received great attention as high-throughput analytic tools in studies of carbohydrate-mediated biological processes. Most of the methods employed to fabricate glycan microarrays rely on the immobilization of modified glycans on the properly derivatized surfaces. This immobilization strategy requires the availability of modified glycans whose syntheses in many cases are time-consuming and difficult. We have developed a simple and direct immobilization technique that involves a one-step, site-specific attachment of diverse unmodified glycans to the hydrazide-derivatized glass surface. To demonstrate the generality of this direct immobilization method, we examined its use for the construction of carbohydrate microarrays containing a variety of glycans. The results of protein and cell-binding experiments indicate that the glycan microarrays, prepared by using this methodology, are applicable to the rapid evaluation of glycan-mediated biomolecular interactions and the determination of quantitative binding affinities between carbohydrates and proteins.