Acoustic microscopy is expected to be a powerful tool for observing biological matters without chemical staining. We have proposed a new method for two-dimensional acoustic impedance imaging for biological tissue that can perform micro-scale observation without preparing a sliced specimen. A tissue was attached on a 0.5 mm-thick plastic substrate. An acoustic pulse was transmitted from the rear side of the substrate. The reflection intensity was interpreted into local acoustic impedance of the target tissue. In the previous report, we demonstrated the outline of the system and the result of preliminary observation, showing its feasibility. This report deals with the optimization of the observation method, and characterization of the tissue of developing cerebellum. The result shows that change in acoustic impedance of each cerebellar layer depending on postnatal day was correspond to change of structure with growth.