This study investigated the formation of self-assembled binary colloidal crystal (BCC) monolayers using evaporation induced confined area assembly (EICAA), and fabricated a family of various BCCs for cell culture. A library of various BCCs with different structures was established and it was demonstrated that after stabilisation the BCCs have potential to be used as substrates with well-ordered surface topographies and chemistries for manipulating cell-surface interactions. Three cell types including MG63 osteoblasts, L929 fibroblasts, and primary human adipose-derived stem cells (hADSCs) show different responses on selected surfaces (either between BCCs or BCCs vs. flat controls). In general, cell spreading was inhibited on BCCs due to surface topography. However, the chemical composition presented on the BCCs can compensate the topographic effect depending on what combination was used. The ordered topographic and heterogeneous chemical patterns provide a complexity of surface properties and have potential to be selectively modified with desired biomolecules for controlling biointerface interactions.