The picosecond ultrasonics technique is used to investigate the viscoelastic properties of nucleus of fixed single osteoblast progenitor cells adhering on a titanium alloy substrate. A two-color probing picosecond ultrasonics and a fluorescence visualization setups were developed and combined to allow to distinguish subcomponents inside the cell under investigation. It opens the way for quantitative measurements of the viscoelastic properties of single cells and of their sub-micrometer thickness. It is shown that a blue probe, λ = 400 nm, is preferable to a red probe, λ = 800 nm, to perform these measurements with fixed sub-micrometer bone cells. 26 GHz acoustic frequencies are detected in cells as thin as 135 nm. A 1D analytical model of the acoustic generation and of the optical detection is used to describe the experimental results. The nucleus longitudinal elastic moduli (13–16 GPa) and dynamic viscosities (13–30 cP) are measured at high frequencies (GHz) from a time-frequency analysis of the experimental data of fixed single cells.