A biosensor formed by a combination of silicon (Si) micropore and graphene nanohole technology is expected to act as a promising device structure to interrogate single molecule biopolymers, such as deoxyribonucleic acid (DNA). This paper reports a novel technique of using a focused ion beam (FIB) as a tool for direct fabrication of both conical-shaped micropore in Si3N4/Si and a nanohole in graphene to act as a fluidic channel and sensing membrane, respectively. The thinning of thick Si substrate down to 50 µm has been performed prior to a multi-step milling of the conical-shaped micropore with final pore size of 3 µm. A transfer of graphene onto the fabricated conical-shaped micropore with little or no defect was successfully achieved using a newly developed all-dry transfer method. A circular shape graphene nanohole with diameter of about 30 nm was successfully obtained at beam exposure time of 0.1 s. This study opens a breakthrough in fabricating an integrated graphene nanohole and conical-shaped Si micropore structure for biosensor applications.