Lilla Estelle Appleton was born in Victory, Vermont, on November 9, 1858, to George Ashley Appleton (1823-1913) and Fanny Reed (Wooster) Appleton (1833-1918). In 1875 she began teaching in rural schools in Vermont, and she studied at the State Normal School in Randolph, Vermont, graduating in 1879.

In 1881 she and her brother John Ashley Appleton (1856–83; enr. 1881-83) entered Oberlin College, joining their brothers Fayette Gilman Appleton (1854-1938; B.D. 1884) and George Stebins Appleton (1862-1941; enr. 1880-83). After one year as a student in the Preparatory Department, Lilla Estelle Appleton entered the Literary Course and graduated with the L.B. in 1886.

Following her graduation Lilla Estelle Appleton returned to teaching. She taught at Kawaiahao Seminary in Honolulu, Hawaii, from 1886 to 1888. During 1888-89 she taught in Lake Henry, South Dakota, and then spent three months in 1889 teaching at Santee Indian Training School in Santee Agency, Nebraska.

She returned to Oberlin College in 1889 for further study in the Philosophical Course. She received the Ph.B. in 1890 and subsequently spent a year (1890-91) studying in the Oberlin Conservatory of Music.

Lilla Estelle Appleton returned to Hawaii in 1891 and taught in government schools there for the next three years. She returned to Vermont in 1895-96 as a teacher and lecturer.

She subsequently spent two years at the Oswego (New York) State Normal School, pursuing studies in the classical (1897) and critic courses (1898). Following her graduation from these courses, Lilla Estelle Appleton began teaching in teacher-training courses. Over the next few years she taught and was a school administrator at State Normal Schools in South Dakota, Minnesota, and Washington.

In 1903, Lilla Estelle Appleton resigned her position as principal of the City Normal Training Class in Kalamazoo, Michigan, to enter into graduate studies at the University of Chicago. At the University of Chicago, Lilla Estelle Appleton earned the Masters of Philosophy (Ph.M.) degree in 1903, the Masters of Science (S.M.) degree in 1904, and the Doctor of Philosophy (Ph.D.) degree in 1909.

Between 1907 and 1916, she served as head of teacher training programs at Upper Iowa University (Fayette, Iowa), Marshall College (Huntington, West Virginia), Milwaukee-Downer College (Milwaukee, Wisconsin), and at the Kindergarten Training School in Grand Rapids, Michigan.

Throughout her life, Lilla Estelle Appleton continued to pursue her studies. She was a senior research fellow at Clark University, 1908-09, and also conducted research at Columbia University in 1910-11. She pursued courses studying mental testing at The Training School in Vineland, New Jersey, in 1914 and 1916.

In 1919, after three years of doing primarily editorial work, Lilla Estelle Appleton took the position of Head of the Department of Psychology and Education at Oxford College for Women in Oxford, Ohio. When that institution was closed in 1928, she retired from teaching and returned to Chicago where she did research work at the University of Chicago, revising and expanding her doctoral dissertation. She was the author of many published articles, mostly in the area of her chief interest, child study.

In 1911 she was invited to join the Institut Solvay – Institut de Sociologie, an international organization for scientific research work, based in Brussels, Belgium. She was also honored by the American Association for the Advancement of Science (A.A.A.S.) which named her a Fellow in 1915.

Lilla Estelle Appleton died of abdominal cancer in Chicago on May 8, 1937. She was buried in Granby, Vermont.