The objective of this study was to obtain quantitative data on the dependence of reflected ultrasound signal on the signal incident angle in the enamel layer of the extracted human incisors. The measurements were conducted in vitro on freshly extracted human mandibular incisors by using a high-frequency focused ultrasonic transducer (PT50-3-6, Toray Engineering, Japan) in pulse-echo mode. The angle dependences of the enamel thickness measurements were studied with high accuracy using a transducer swing mechanism of the scanning acoustic microscope (Tessonics AM1103, Windsor, ON, Canada). Similar angular measurements were carried out by using the same PT50-3-6 transducer integrated into a compact hand-held probe to mimic a real dental procedure. The received thickness results were compared with the ones obtained through direct optical microscopy measurements of the teeth cross-section exposed by cutting the incisors down their long axis. Obtained results showed a close correlation between ultrasonic and optical microscopy measurements of the dental enamel thickness. The estimated difference in enamel layer thickness measured by the acoustic and optical microscopes did not exceed 12%. The dependence of reflected ultrasound signal on the signal incident angle in the enamel layer of extracted human incisors has been studied. The measurement conditions when the reflected ultrasound signals are not incident angle-dependent have been established.