Dubois was born in Eijsden in the Netherlands and studied medicine at the University of Amsterdam. After briefly working there as a lecturer in anatomy, he served as a military surgeon in the Dutch East Indies, now Indonesia, from 1887 to 1895. On his return to Amsterdam he held the chair of geology, paleontology, and mineralogy from 1899 until his retirement in 1928.

The decision to go to the Indies was no accident. Dubois was determined to find the ‘missing link’ and had reasoned that such a creature would have originated in proximity to the apes of Africa or the orangutan of the Indies. After several years fruitless search in Sumatra, Dubois moved to Java and in 1890 discovered his first humanoid remains (a jaw fragment) at Kedung Brubus. The following year, at Trinil on the Solo river, he found the skullcap, femur, and two teeth of what he was later to name Pithecanthropus erectus, more commonly known as Java man. He published these findings in 1894.

Although Dubois's estimate of the cranial capacity of Pithecanthropus was, at 850 cubic centimeters (later estimates ranged up to 940 cubic centimeters), on the low side for a hominid, the femur it had been found with indicated to Dubois that it must be a form with a very erect posture. However many doubted this, stating the usual objections that the remains belonged to different creatures, to apes or (Rudolf Virchow's view) to deformed humans. So irritated did Dubois become by this reception that he withdrew the fossils from view, keeping them locked up for some 30 years.

When they were again made available to scholars in 1923 and Peking man was discovered in 1926 it at last became widely agreed that Pithecanthropus was, as Dubois had earlier claimed, a link connecting apes and man. By this time however Dubois would have no part of such a consensus and began to insist the bones were those of a giant gibbon, a view he maintained until his death.