It is useful for maturity evaluation of regenerating cartilage to measurethe temporal change of its stiffness. In this study, a laser andhydrophone-based measurement of ultrasound vibration applied to the measuringobject (sample) is investigated for evaluating the stiffness of thinregenerating cartilage sample. Surface particle velocity and acoustic pressureof the sample when applying sinusoidal ultrasound to the sample are measured bya laser Doppler vibrometer and a hydrophone, respectively. Then the acousticimpedance is evaluated by using the measured particle velocity and acousticpressure. The feasibility of this method was investigated by conductingfundamental experiments using agar-based homogeneous phantoms. Moreover, thismethod was also applied to in vitro measurements using auricular cartilagesamples regenerated in beagles.