We have proposed a new method for two-dimensional acoustic impedance imaging for biological tissue that can perform micro-scale observation without slicing the specimen. A tissue was placed on a plastic plate of 0.5 mm in thickness. An acoustic pulse with a frequency range up to 100 MHz was transmitted from the "rear side" of the plate, the acoustic beam being focused at the boundary between the tissue and plate. The reflection intensity was interpreted into local acoustic impedance of the target tissue. An acoustic impedance microscopy with 200 × 200 pixels, its field of view being 2×2 mm, was obtained by mechanically scanning the transducer. Quantification of acoustic impedance was performed using water or an appropriate material as a reference. The accuracy was evaluated using saline with various NaCl content. A rat cerebellum was employed as the specimen. The development of parallel fiber in cerebella cultures was clearly observed as the contrast in acoustic impedance. The proposed technique is believed to be a powerful tool for biological tissue characterization, as neither staining nor slicing is required.