The interaction between microorganisms and nanoparticles is a crucial step towards understanding the subsequent biological effect. In this study, the interaction between TiO2 nanoparticles and bacterial cell membrane was investigated by Two-dimensional Correlation Fourier Transformation Infrared spectroscopy (2D-FTIR-COS) using bacterial ghosts (BGs), which are non-living bacterial cell envelopes devoid of cytoplasm. The synchronous map of 2D-FTIR-COS results indicated that the functionalities in proteins of BGs preferentially interacted with TiO2 nanoparticles; whereas the interaction of TiO2 nanoparticles with characteristic functionality in polysaccharides (COH) and phospholipids (PO) were very weak or insensitive. This conclusion was further corroborated by settling of TiO2 nanoparticles in the presence of pure protein, polysaccharide and phospholipid represented by bovine serum albumin (BSA), alginate and phosphatidylethanolamine (PE). Additionally, the asynchronous map of 2D-FTIR-COS indicated a sequential order of functionalities bonded to TiO2 nanoparticles with the order of: COO− > aromatic CC stretching > NH, amide II > CO, ketone. These findings contribute to deeper understanding of the interaction between TiO2 nanoparticles and bacterial cell membrane in aquatic systems.