This study aims to design interactive e-books and evaluate their effectiveness in students’ study performance. The process of designing interactive e-books based on design principles of content, form, and high applicability using Kotobee Author software has been built and designed for Biology grade 10. Interactive e-books have fully met requirements in terms of supporting online learning with lectures, questions, videos, sharing links, and documents that are suitable for readers' levels and received very good feedback from students. Experimental study designs were used to determine academic achievement and self-study competence. The research sample was 82 grade 10 students divided into two groups, the experimental group learned Biology through interactive e-books, while the control group learned through printed books. The experimental results show that there is a significant difference between the experimental groups in terms of learning outcomes and self-study competence as well as the level of interest of students in Biology lessons when learning by e-books. Interactive e-books can be used in teaching Biology in particular or other subjects in general to improve learning outcomes and self-study competence of students in Vietnam.