A great challenge for nanotechnology is to controllably organize anisotropic nanomaterials into well-defined three-dimensional superstructures with customized properties. Here we successfully constructed anisotropic Au nanorod (AuNR) helical superstructures (helices) with tailored chirality in a programmable manner. By designing the X pattern of the arrangement of DNA capturing strands (15nt) on both sides of a two-dimensional DNA origami template, AuNRs functionalized with the complementary DNA sequences were positioned on the origami and were assembled into AuNR helices with the origami intercalated between neighboring AuNRs. Left-handed (LH) and right-handed (RH) AuNR helices were conveniently accomplished by solely tuning the mirrored-symmetric X patterns of capturing strands on the origami. The inter-rod distance was precisely defined as 14 nm and inter-rod angle as 45 degrees, thus a full helix contains 9 AuNRs with its length up to about 220 nm. By changing the AuNR/origami molar ratio in the assembly system, the average number of AuNR in the helices was tuned from 2 to 4 and 9. Intense chiroptical activities arose from the longest AuNR helices with a maximum anisotropy factor of similar to 0.02, which is highly comparable to the reported macroscopic AuNR assemblies. We expect that our strategy of origami templated assembly of anisotropic chiral superstructures would inspire the bottom-up fabrication of optically active nanostructures and shed light on a variety of applications, such as chiral fluids, chiral signal amplification, and fluorescence combined chiral spectroscopy.