We developed a novel vacuum-assisted microcontact printing (mu CP) process that presents a powerful method for patterning functional materials with precise alignment. The printing pressure of the vacuum-assisted mu CP was applied using the pressure difference between the inside and outside of an elastomeric stamp. A double exposure microfabrication process was adopted for manufacturing different height protrusions on the elastomeric stamps. The outer protrusion was designed to be higher than the printing patterns, thereby acting as a vacuum sealing wall. The printing pressure was easily applied and controlled using commercial syringes and motorized syringe controllers. A high printing pressure exceeding 10 psi was applied uniformly to the target substrate. Precision alignment was realized using a common optical alignment system. During the alignment process, damage to the previously patterned material and undesired printing patterns due to stamp dragging were avoided by imposing a separation distance between the printed pattern and the substrate. Several functional materials, including proteins and nanomaterials, could be successively patterned. Protein-protein, protein-nanowire, and three-dimensionally patterned nanowires are described. This versatile vacuum-assisted mu CP process is a powerful means for implementing the large-scale fabrication in bio- and nano-technologies and related applications.