The development of supramolecular hydrogel scaffolds for the precise positioning of biochemical cues is paramount for applications such as tissue engineering. Nucleic acid engineering allows fabrication of three-dimensional (3D) nanostructures with high variability and nanoscale precision. In this study, aptamers were anisotropically functionalized onto branched DNA nanostructures to control their cell adhesion capability, and their efficiency as biological signal inducers for 3D cell cultivation was investigated. Each arm of the X-shaped DNA nanostructure (X-DNA) was functionalized with photo-cross-linkable or cell adhesion moieties, and the steric hindrance of the 3D DNA nanostructures on a cell was optimized. X-DNA nanostructures with cell-positioning parameters were rapidly photopolymerized to form hybrid hydrogels, and their effects on cell behaviors and positions were investigated. We observed that aptamer-functionalized X-DNA nanostructures exhibited significantly enhanced cell proliferation and provided homogeneous distribution and target-specific adhesion of encapsulated cells within hydrogel matrices. Overall, the anisotropic functionalization of DNA nanostructures provides a controllable function for the advancement of conventional 3D culture platforms.