Understanding the interaction between graphene oxide (GO) and a lipid membrane is significant for exploring the biocompatibility and cytotoxicity of GO, which is the basis for utilizing GO in the fields of biosensors, bioimaging drug delivery, antibacterials, and so on. In this article, we monitored the dynamic process of the morphology change and detachment of lipid bilayers on mica substrates prompted by GO sheets by in situ atomic force microscope (AFM) imaging. It was found that the bare lipid bilayer dramatically expanded in height and would be unstable and detachable from the mica substrates as induced by GO. The detached lipid molecules were found to bind to the GO surface. The results also imply that GO is likely to influence the height and stability of the supported lipid bilayers (SLBs) by adsorbing metal ions such as calcium ions that were used to stabilize the bilayer structures on the mica substrate. These findings illustrate a complicated effect of GO on the SLBs and should be helpful in future applications of GO in biotechnology.