By means of scanning tunneling microscopy (STM), we succeeded for the first time in the quantitative analysis of the intramolecular conformation of a supramolecule, cyclodextrin (CyD) necklace, driven by hydrogen bonding. Contrary to the current model, based on macroscopic analyses, which indicates that all CyDs are arranged in head-to-head or tail-to-tail (secondary-secondary or primary-primary hydrogen bonding) conformation, about 20% head-to-tail (primary-secondary hydrogen bonding) conformation was found to exist in the molecule. In addition, comparing the STM results with the theoretical model of the necklace formation, the formation ratio of the tail-to-tail and head-to-tail conformations due to the strength difference between primary-primary and primary-secondary hydrogen bonds of CyDs was directly obtained, for the first time, to be 2:1.