We developed a new, advanced, simple and non enzymatic approach for the colorimetric detection of glucose based on calix[4]arene/phenyl boronic acid (CX-PBA)functionalized gold nanoparticles (AuNPs). This molecular receptor proficiently and selectively recognizes glucose due to its ability to reversibly bind diol-containing compounds. The assembly was characterized by transmission electron micrograph (TEM), dynamic light scattering (DLS), UV-Vis, FT-IR, ESI-MS and (1)H NMR spectrometry, which demonstrates the binding affinity for glucose via a boronic acid-diol interaction. The linear range for glucose was found to be 5-100 nM with phosphate buffer pH 10, with a lower detection limit of 4.3 nM. Interference by other saccharides was negligible. The biosensor has been successfully applied to estimate the glucose in human blood serum samples and the results compared well to an automatic analyzer. With the advantages of high sensitivity, selectivity and low sample volume, this method is potentially suitable for the on-site monitoring of glucose.