Understanding of bio-nano-interfaces of living mammalian cells will benefit the identification of cellular alterations (e.g. nucleic acids, amino acids, biomechanics, etc.) due to external stimuli, the design of biomaterials (e.g. nanoparticles, nanotubes) and the investigation of the interaction between cells and bio-nano-interfaces (e.g. cell differentiation on 3D nanostructured materials). Analytical techniques can be applied to evaluate the chemical, physical, and mechanical properties of mammalian cells when exposed to such bio-nano-interfaces. In this study, non-invasive advanced spectroscopy techniques including atomic force microscopy (AFM) and Raman microscopy (RM), in conjunction with traditional biological methods are utilized to elucidate specific characteristic information for biological samples and how these property changes reflect the interaction with external stimuli. The focus of this dissertation is on the biophysical, biochemical and cytotoxic detection of mammalian cells interacting with bio-nano-interfaces, and this dissertation can be classified into three topics: biomechanics/cellular biopolymers measurement, bio-interfaces and nano-interfaces studies. For the topic of biomechanics/cellular biopolymers measurement, cellular biophysical and biomechanical properties could be used as differentiation markers to classify cellular differentiation. For the bio-interfaces part, it was observed that BRMS1 expression changed cellular biochemical and biomechanical properties, and the expressions of reactive oxidative species (ROS), apoptosis and cell viability of five types of cells displayed similar patterns over doxorubicin (DOX) incubation time. Secondly, A549 cells were treated with diesel exhaust particles (DEP) and resveratrol (RES) to study the effect of RES on the DEP-induced cells, and it was found that RES can alleviate DEP intervention on cellular structure and increase DEP-induced biomechanical and inflammatory changes. For the nano-interfaces topic, first we synthesized a hybrid nanoparticle with the multimodal properties of fluorescence imaging, Surface-enhanced Raman spectroscopy (SERS) detection and photothermal therapy (PTT) for single living cell analysis of epidermal growth factor receptor (EGFR) and specifically killed cancer cells with high EGFR expression. Additionally, to increase surface area, light-heat conversion efficiency and biocompatibility, we developed a silica coated nanoparticle conjugated with anti-human epidermal growth factor receptor 2 (HER2) antibody. Finally, three-dimensional TiO2 nanotubes with Au nanoparticles coating were synthesized and used to study trophoblast-derived stem-like cells growth on such 3D nanostructures.