Provided is a three-dimensional flash memory including a substrate, a stack structure, a stop layer, two slit trenches, a plurality of vertical channel structures, and a plurality of slit holes. The stack structure is disposed on the substrate. The stack structure includes a plurality of dielectric layers and a plurality of conductive layers stacked alternately. The stop layer is disposed between the substrate and the stack structure. The two slit trenches penetrate through the stack structure to expose the stop layer. The vertical channel structures are disposed between the two slit trenches and penetrate through the stack structure and the stop layer. The slit holes are discretely disposed between the vertical channel structures, and penetrate through the stack structure to expose the stop layer. A method of forming the three-dimensional flash memory is also provided.