• Two-dimensional ice-like water layers form on self-assembled monolayers supporting Cytochrome C under ambient conditions without nanoconfinement.
• Direct characterization of morphology, composition, melting, and crystallization of 2D ice-like water using AFM and nano-AFM-IR.
• The formation is attributed to the activation energy for water desorption from Cyt C being nearly twice that from SAMs.
• Provides a new model system to study water–protein interactions and ice phase transitions under normal conditions.