The blood–brain barrier (BBB) possesses an intricate structure and set of characteristics that effectively restricts the transportation of substances to the central nervous system. Consequently, the development of pharmaceuticals for brain disorders poses significant hurdles. Numerous attempts have been made to produce innovative simulates of BBB that exhibit a striking resemblance to BBB and are suitable for efficient drug screening, as well as being user‐friendly and easily reproducible. One of the models available is the commercial Transwell® system. In this system, chosen endothelial cells form a single layer on the rigid membrane of the upper chamber. In the present study, the two‐dimensional structure of this membrane was exchanged with a three‐dimensional membrane using the 3D printing method and developed a novel approach to fabricate the scaffold in one step on the fabricated Transwell. This novel method of horizontal electrospinning has reduced the mechanical damage associated with conventional vertical electrospinning, resulting in suitable mechanical properties for cell culture. The lower diameter of the nanofibers, the thickness of the mesh, bulk Young's modulus and hydrophobicity compared to conventional mesh‐based counterparts are characteristics which make it more similar to the natural basement membrane and improve cell culture necessities on the scaffold. The designed BBB model was characterized via cell morphology, trans‐endothelial electrical resistance, and permeability value. This human BBB model formed robust cell integrity and achieved the acceptable range of TEER and low permeability that confirm the polyacrylonitrile (PAN) scaffold as an in vitro cell culture platform with promising cell culture results of the brain microvascular endothelial cell line hCMEC/D3.