Biomechanical properties are potential information for the diagnosis of corneal pathologies. An ultrasound indentation probe consisting of a load cell and a miniature ultrasound transducer as indenter was developed to detect the force–indentation relationship of the cornea. The key idea was to utilize the ultrasound transducer to compress the cornea and to ultrasonically measure the corneal deformation with the eyeball overall displacement compensated. Twelve corneal silicone phantoms were fabricated with different stiffness for the validation of measurement with reference to an extension test. In addition, fifteen fresh porcine eyes were measured by the developed system in vitro. The tangent moduli of the corneal phantoms calculated using the ultrasound indentation data agreed well with the results from the tensile test of the corresponding phantom strips (Symbol). The mean tangent moduli of the porcine corneas measured by the proposed method were 0.089 ± 0.026 MPa at intraocular pressure (IOP) of 15 mmHg and 0.220 ± 0.053 MPa at IOP of 30 mmHg, respectively. The coefficient of variation (CV) and intraclass correlation coefficient (ICC) of tangent modulus were 14.4% and 0.765 at 15 mmHg, and 8.6% and 0.870 at 30 mmHg, respectively. The preliminary study showed that ultrasound indentation could be applied to the measurement of corneal tangent modulus with good repeatability and improved measurement accuracy compared to conventional surface displacement-based measurement method. The ultrasound indentation can be a potential tool for the corneal biomechanical properties measurement in vivo.