Engineered surface topographies at the micro- and nanoscale have been shown to effectively regulate physical behavior and biochemical signaling of mechanically sensitive cells, e.g., bone-related cells, especially in 3D. However, scalable and cost-effective methods to fabricate biologically instructive 3D silicon structures remain limited. The deep reactive ion etching (DRIE) process, although widely used in microelectromechanical systems, has not been fully explored for constructing biomimetic substrates that support in vitro bone-related cell cultures. By extending the application of DRIE beyond conventional micromechanical uses, this study introduces a scalable and tunable strategy for constructing functional micro–nano structures suitable for tissue engineering and biointerface research. Several designs and fabrication protocols for a variety of 3D topographic structures, including obelisks, scalloped pillars, and wine glass shapes were developed. Wafer-scale fabrication was achieved with the DRIE technique, particularly with high controllability at micro- and nanoscale for structural height, dimension, and sharpness. By adapting the classic DRIE process to fabricate silicon microstructures with tailored topographies, the resulting approach offers new possibilities for designing and fabricating 3D in vitro cell culture scaffolds, which can be applied for in vitro biomechanical studies, such as cell-chip coupling, cellular guidance, and differentiation.