Provided is a method of forming a nanogap pattern of a biosensor. First, an oxide layer is formed on a substrate and a first nitride layer is formed on the oxide layer. The first nitride layer is partially etched to form a first nitride layer pattern having a first gap that gradually narrows from a top portion to a bottom portion thereof and exposes the oxide layer. A second nitride layer is formed along the first nitride layer and along sidewalls and a bottom surface of the first gap. The second nitride layer is etched to form a second nitride layer pattern having a second gap narrower than the first gap on the sidewalls of the first gap. The oxide layer is etched by using the second nitride layer pattern as an etching mask to form an oxide layer pattern having a third gap, and thus, the nanogap pattern is completed.