Wilkin was born on March 30, 1904 in Rochester, New York, the third of four children. At age 2, Eloise moved with her family to New York City, but spent every summer with her siblings at a relative's home in western New York State. Memories forged there of family togetherness and the outdoors would influence her famous illustrations of nature, children, and family life. Wilkin won a drawing contest for New York schoolchildren at age 11 and graduated from the Rochester Athenaeum and Mechanics Institute, now the Rochester Institute of Technology, in 1923.
Soon after college graduation, Eloise and friend Joan Esley opened an art studio in Rochester, NY but struggling to find work, the pair moved to New York City, where Century Company gave Eloise her first book to illustrate, The Shining Hours. Many of her early illustrations were for school books. Early in her career Eloise illustrated paper dolls for Samuel Gabriel & Sons, Playtime House and Jaymar. Wilkin often illustrated the titles of her sister, children's author Esther Burns Wilkin, who married Eloise's brother-in-law. The first of the Wilkins' collaborations was Mrs. Peregrine and the Yak, published by the Henry Holt Company.
In 1944, Wilkin signed an exclusive contract with original Little Golden Books publisher Simon & Schuster requiring her to illustrate three books each year. She often used her children and grandchildren and their friends as models for her illustrations. A devout Christian, Wilkin frequently illustrated religious picture books including several compilations of prayers for children.

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