Intracytoplasmic sperm injection (ICSI) is an infertility treatment where a single sperm is immobilised and injected into the egg using a glass injection pipette. Minimising vibration in three orthogonal axes is essential to have precise injector motion and full control during the egg injection procedure. Vibration displacement sensing using physical sensors in ICSI operation is challenging since the sensor interfacing is not practically feasible. This study proposes a non-invasive technique to measure the three-dimensional vibrational motion of the injection pipette by a single microscope camera during egg injection. The contrast-limited adaptive histogram equalization (CHALE) method and blob analyses technique were employed to measure the vibration displacement in axial and lateral axes, while the actual dimension of the focal axis was directly measured using the Brenner gradient algorithm as a focus measurement algorithm. The proposed algorithm operates between the magnifications range of 4× to 40× with a resolution of half a pixel. Experiments using the proposed vision-based algorithm were conducted to measure and verify the vibration displacement in axial and lateral axes at various magnifications. The results were compared against manual procedures and the differences in measurements were up to 2% among all magnifications. Additionally, the effect of injection speed on lateral vibration displacement was measured experimentally and was used to determine the values for egg deformation, force fluctuation, and penetration force. It was shown that increases in injection speed significantly increases the lateral vibration displacement of the injection pipette by as much as 54%. It has been demonstrated successfully that visual sensing has played a key role in identifying the limitation of the egg injection speed created by lateral vibration displacement of the injection pipette tip.