Mussel-inspired two-dimensional freestanding, alkyl-polydopamine (alkyl-PDA) Janus nanosheets, with a well-controlled nanometer thickness and a lateral size of up to micrometers, have been developed. A self-assembled octadecylamine (ODA) bilayer is used as the reactive template for the dopamine polymerization, resulting in the formation of well-defined nanosheets. The alkyl-PDA nanosheets show an amphiphilic nature with hydrophilic PDA and hydrophobic alkyl chains on opposing sides. The nanosheets can be used to functionalize many substrates and is dependent on the configuration of surface of the nanosheets. The nanosheets are quite stable, as the morphology is preserved after carbonization at 900 °C. Post-modification of the nanosheets can be easily achieved because of the reactive nature of PDA. This work will provide a new strategic approach for fabricating polymeric Janus nanosheets, which can find applications for surface modifications, catalyst supports, and guided self-assembly.