Abstract: Precise control over the assembly of anisotropic plasmonic gold nanostructures with relative spatial directionality and sequence asymmetry remains a major challenge and offers great fundamental insight and optical application possibilities. Here, a novel strategy is developed to anisotropically functionalize gold nanorods (AuNRs) by using a DNA-origami-based precise machine to transfer essential DNA sequence configurations to the surface of the AuNRs through an intentionally designed toehold-initiated displacement reaction. Different AuNR products are examined via hybridization with DNA-AuNPs that display distinct elements of regiospecificity. These assembled anisotropic plasmonic gold nanostructures based on the DNA-origami precise machine inherit the encoded information from the parent platform with high fidelity and show fixed orientation and bonding anisotropy, thereby generating discrete plasmonic nanostructures with enhanced Raman resonance.