Abstract Surface structure of trehalose dihydrate crystals in liquid was investigated by using high-resolution frequency modulation atomic force microscopy (FM-AFM). While trehalose exhibits remarkable bioprotective properties, its molecular-scale surface structure under liquid conditions remains poorly understood. Crystals prepared from supersaturated aqueous solution were observed in hexanol, a non-solvent that can suppress dissolution and enables stable imaging. Wide-area imaging revealed flat terraces emerged through gradual dissolution of surface microcrystals. The measured step height of 0.6–0.7 nm corresponds to a single molecular layer, indicating that the observed surface is the (100) plane. High-resolution images exhibited periodic structures with spacings consistent with crystallographic lattice parameters. Two distinct contrast patterns were identified, reflecting two types of trehalose molecule with different orientation in the unit cell. These findings demonstrate the feasibility of stable liquid-phase AFM imaging of trehalose crystal and provide direct insights into its surface molecular arrangement, contributing to understanding of their physicochemical functionality.