Two-dimensional gold-assembled nanomaterials have garnered considerable interest in biomedical applications such as wearable sensors and flexible devices. The assembly can be accomplished via attractive interactions between gold nanoparticles (GNPs) and a standard polymer. It is highly desirable and a great challenge to develop a new assembly method that can provide formed materials with novel properties. In this work, we explored complementary DNA-functionalized gold nanoparticles (DNA-GNPs) as building blocks to establish multifunctional two-dimensional gold nanomaterials via layer-by-layer (LBL) assembly. We found that the DNA-mediated assembly endows GNP films with a superlattice and their sonic behavior could be regulated in a controllable manner through altering the sequence of SH-DNA and linker DNA, which was detected by the electric-induced ultrasound method. Our observation suggested that this DNA-mediated assembly of GNP films with controllable sonic behaviors could greatly promote the biomedical application of two-dimensional assembled-gold nanomaterials.