Directly correlating the morphology and composition of interfacial water is vital not only for studying water icing under critical conditions but also for understanding the role of protein-water interactions in bio-relevant systems. In this study, we present a model system to study two-dimensional (2D) water layers under ambient conditions by using self-assembled monolayers (SAMs) supporting the physisorption of the Cytochrome C (Cyt C) protein layer. We observed that the 2D island-like water layers were uniformly distributed on the SAMs as characterized by atomic force microscopy, and their composition was confirmed by nano-atomic force microscopy-infrared spectroscopy and Raman spectroscopy. In addition, these 2D flakes could grow under high-humidity conditions or melt upon the introduction of a heat source. The formation of these flakes is attributed to the activation energy for water desorption from the Cyt C being nearly twofold high than that from the SAMs. Our results provide a new and effective method for further understanding the water-protein interactions.