DNA-based nanomaterials are gaining popularity as uniform and programmable bioengineering tools as a result of recent solutions to their weak stability under biological conditions. The DNA nanotechnology platform uniquely allows decoupling of engineering parameters to comprehensively study the effect of each upon cellular encounter. We here present a systematic analysis of the effect of surface parameters of DNA-based nanoparticles on uptake in three different cell models: tumor cells, macrophages, and dendritic cells. The influence of surface charge, stabilizing coating, fluorophore types, functionalization technique, and particle concentration employed is found to cause significant differences in material uptake among these cell types. We therefore provide new insights into the large variance in cell type-specific uptake, highlighting the necessity of proper engineering and careful assay development when DNA-based materials are used as tools in bioengineering and as future nanotherapeutic agents.