Robert W. Landis was born in Philadelphia in 1809 of pious Baptist parents. At age 17 he made a profession of religion, joined the Baptist Church and began his theological training. He, however, received little formal education, attending the Manual Labor Academy of Germantown, Pennsylvania, for fifteen months and being tutored for three months by an Episcopal clergyman. All his remarkable scholarly attainments were gained by personal study. At age 20 he joined the Presbyterian Church, which licensed him in 1831 and ordained him the next year. He served as pastor or supply pastor, 1831-1861, 1865-1868, in Pennsylvania, New York, Michigan, Kentucky and Delaware. During the Civil War he served three and a half years in the Union Army as chaplain of a regiment of cavalry. In 1868-1869 he occupied Dr. Robert J. Breckinridge's chair in the Presbyterian Theological Seminary at Danville, Kentucky, and later accepted a permanent professorship at the seminary.

Landis early displayed polemic skill and was engaged in controversy throughout his life. At 21 he published his first book, on the Trinity, to combat an "Arian" who had challenged him to a debate. Some of his theological views being at variance with leading members of the Presbytery of Philadelphia, he went to New York. Challenged by followers of Alexander Campbell, he engaged in public discussions, lectured, and published articles on Campbellism, including his "Rabbah Taken." During his pastorate at Bethlehem, New Jersey, he was opposed by his predecessor and the distillers, was attacked in Presbytery, hauled into civil court and fined. His book on the resurrection was written in reply to a book of Professor George Bush, a Presbyterian clergyman and scholar who became a Swedenborgian. Landis' manuscript treatise on the doctrine of imputation was written in opposition to the theology of Dr. Charles Hodge of Princeton Theological Seminary. Landis published theological and exegetical articles in various periodicals including The Danville Quarterly Review.

Landis collected a large and valuable library containing many rare books on theological and related subjects. Intending to give the library to Union Theological Seminary in New York if they would publish his work on imputation, he eventually gave it to Central University, Richmond, Kentucky, which agreed to comply with his stipulation but probably never did.