Mohorovičić was born the son of a shipwright in Volosko, Croatia, and educated at the University of Prague. He worked initially as a teacher and at the meteorological station in Bakar before being appointed a professor at the Zagreb Technical School in 1891 and at Zagreb University in 1897.

In 1909 he made his fundamental discovery of the Mohorovičić discontinuity (or Moho). From data obtained while he was observing a Croatian earthquake in 1909, Mohorovičić noticed that waves penetrating deeper into the Earth arrived sooner than waves traveling along its surface. He deduced from this that the Earth has a layered structure, the crust overlaying a more dense mantle in which earthquake waves could travel more quickly. The abrupt separation between the crust and the mantle Mohorovičić calculated as being about 20 miles (32 km) below the surface of the Earth.

As the crust is much thinner under the ocean beds – in some places only 3 miles thick – a project was set up in the 1960s to drill through the crust to the mantle. Mohole, as it became known, failed, however, largely as a result of the great financial cost involved and the inadequate technological expertise available for such a project.