Endothelial cells (ECs) line the inner walls of blood vessels, respond to shear stress by elongating in the direction of flow. Engineering aligned ECs in vitro is essential for modeling human vascular diseases and for drug testing. Current microfluidic approaches mainly rely on unidirectional laminar flow, uniform coating of surfaces to improve cellular adhesion or alteration of the surface topography. Challenges persist due to shear stress‐induced changes in cellular behavior, especially in complex multicellular environments and the time needed for the cells to align and polarize inside the microfluidic conduits. Generally, protein coating processes and physical treatments are also not compatible with the steps required for the assembly of microfluidic devices. This approach employs aerosol jet printing (AJP) to precisely pattern poly(3,4‐ethylenedioxythiophene) polystyrene sulphonate (PEDOT:PSS) within microfluidic chambers in a single step. It is shown that the PEDOT:PSS is biocompatible and facilitates EC adhesion, patterning, elongation, and alignment. Under capillary flow, the cells retain their pattern‐induced morphology over 7 d, confirming the efficacy of the approach in promoting cellular organization, eliminating the need for external pumps. Furthermore, it is demonstrated that the PEDOT:PSS pattern retains structural integrity and electrical stability following oxygen plasma treatment, required for assembling of fully enclosed microfluidic devices.