Scalable preparation of nanoparticle assemblies and superstructures with control over the size, composition, and geometry represents a challenge for many practical applications of nanomaterials. DNA nanotechnology offers both versatility of particle arrangements and multiple gateways to interesting optical, electronic, biological, and catalytic properties. In this work we used a polymerase chain reaction (PCR) to produce complex superstructures from heterogeneously-sized gold nanoparticles. By controlling the number of PCR cycles and primer density, two distinct superstructures were assembled at asymmetric and symmetric PCR. With the increasing of PCR number, the size of structures was accordingly increased in the range 35–600nm. More importantly, the scaled-up GNP superstructures exhibited tunable chirality, which showed distinct differences between asymmetric and symmetric PCR. The origin of the chirality was analyzed from both an experimental and theoretical point of view. The heterogeneity of GNPs and conformational transition of self-assembly contributed to the occurrence of chirality in the GNP superstructures. PCR could be a very important and potentially useful tool for the controllable preparation of scaled-up superstructures with tunable chirality, and this experimental and mechanical research could significantly promote the technical development of chiral material preparation.