Heuglin was born in Hirschlanden (now part of Ditzingen) in Württemberg. His father was a Protestant pastor, and he was trained to be a mining engineer. He was ambitious, however, to become a scientific investigator of unknown regions, and with that object studied the natural sciences, especially zoology.
In 1850 he went to Egypt where he learnt Arabic, and visited the Red Sea and Sinai. In 1852 he accompanied Dr. Christian Reitz, Austrian consul at Khartoum, on a journey to Ethiopia, and after Reitz's death was appointed his successor in the consulate. While he held this post he travelled in Ethiopia and Kordofan, making a valuable collection of natural history specimens. In 1857 he journeyed through the coast lands of the African side of the Red Sea, and along the Somali coast.
In 1860 he was chosen as leader of an expedition to search for Eduard Vogel, his companions including Werner Munzinger, Gottlob Kinzelbach, and Hermann Steudner. In June 1861 the party landed at Massawa, having instructions to go direct to Khartoum and then to Ouaddai, where Vogel was thought to be detained. Heuglin, accompanied by Hermann Steudner, made a wide detour through Abyssinia and the Galla country, and in consequence the leadership of the expedition was taken from him. He and Steudner reached Khartoum in 1862 and there joined the party organized by Alexandrine Tinné and her mother Henriette Tinne-van Capellen, who then had just returned from a White Nile journey to Gondokoro. With both women and on their own account they explored a great part of the Bahr-el-Ghazal, where Steudner died of fever on 10 April 1863 and Alexine's mother on 20 July.
After having reached Cairo with Alexandrine Tinne, Heuglin returned to Europe in February 1864. In 1870 and 1871 he made a valuable series of explorations in Spitsbergen and Novaya Zemlya; but 1875 found him again in north-east Africa, in the country of the Beni, Amer and northern Abyssinia. He was preparing for an exploration of the island of Socotra, when he died in Stuttgart. It is principally by his zoological, and more especially his ornithological, labours that Heuglin has taken rank as an independent authority.