Characterizing the mechanical characteristics of living cells and cell–biomaterial composite is an important area of research in bone tissue engineering. In this work, an in situ displacement-controlled nanoindentation technique (using Hysitron Triboscope) is developed to perform nanomechanical characterization of living cells (human osteoblasts) and cell–substrate constructs under physiological conditions (cell culture medium; 37 °C). In situ elastic moduli (E) of adsorbed proteins on tissue culture polystyrene (TCPS) under cell culture media were found to be ∼4 GPa as revealed by modulus mapping experiments. The TCPS substrates soaked in cell culture medium showed significant difference in surface nanomechanical properties (up to depths of ∼12 nm) as compared to properties obtained from deeper indentations. Atomic force microscopy (AFM) revealed the cytoskeleton structures such as actin stress fiber networks on flat cells which are believed to impart the structural integrity to cell structure. Load-deformation response of cell was found to be purely elastic in nature, i.e., cell recovers its shape on unloading as indicated by linear loading and unloading curves obtained at 1000 nm indentation depth. The elastic response of cells is obtained during initial cell adhesion (ECell, 1 h, 1000 nm = 4.4–12.4 MPa), cell division (ECell, 2 days, 1000 nm = 1.3–3.0 MPa), and cell spreading (ECell, 2 days, 1000 nm = 6.9–11.6 MPa). Composite nanomechanical responses of cell–TCPS constructs were obtained by indentation at depths of 2000 nm and 3000 nm on cell-seeded TCPS. Elastic properties of cell–substrate composites were mostly dominated by stiff TCPS (EBulk = 5 GPa) lying underneath the cell.