The acoustic microscope is a powerful tool for the observation of biological matters. Non-invasive in-situ observation can be performed without any staining process. Acoustic microscopy is contrasted by elastic parameters like sound speed and acoustic impedance. We have proposed an acoustic microscope that can acquire three-dimensional acoustic impedance profile. The technique was applied to cell-size observation. Glial cells were cultured on a 70 μm-thick polypropylene film substrate. A highly focused ultrasound beam was transmitted from the rear side of the substrate, and the reflection was received by the same transducer. An acoustic pulse, its spectrum spreading briefly 100 through 450 MHz, was transmitted. By analyzing the internal reflections in the cell, the distribution of characteristic acoustic impedance along the beam direction was determined. Three-dimensional acoustic impedance mapping was realized by scanning the transducer, exhibiting the intra-cellular structure including nucleus and cytoskeleton.