Understanding the effect of humidity on permeability of Graphene oxide (GO) membranes is vital for their use in membrane based water filtration. Membrane based water filtration is an efficient method for addressing concerns of water scarcity and security as they can provide a low impact method of filtration without reliance on chemical additives or phase changes. GO membranes are formed by the assembly of GO sheets during the drying process, which could be impacted by the humidity of the drying environment. As upstate New York features a humid subtropical variety throughout the year, the ambient humidity changes abruptly even in a controlled ambient lab condition, which could impact the quality of the GO membrane fabricated throughout the year. A relative humidity variation from 10 RH% Jan to 79 RH% July at 21oC Lab condition was identified during the GO membrane preparation process from Jan to Aug 2023. Given the unique nature of GO membranes, it is important to understand how humidity affects the characteristics of the membranes themselves in order to better design GO membrane fabrication. In this work, permeation of ethanol and water vapor molecules through GO membranes formed at three different relative humidity levels were studied and analyzed with Fick’s first Law to understand how relative humidity affects the permeation channel of the GO membrane. These GO membranes are further characterized with contact angle, X-ray diffraction spectrum, and fourier-transform infrared spectroscopy to reveal the morphology and structure difference caused by humidity of the environment. These results will provide an in depth understanding about how the environmental humidity affects the permeation channel of GO membranes.