The purpose of this research was to determine (1) the effect of e-portfolio use on understanding Biology concepts of students at different academic levels and (2) knowing student responses in the use of e-portfolios. This research is experimental research using a one-shot case study design. Data on understanding students' concepts were collected through the results of learning tests, while student response data were collected through questionnaires. The data analysis used parametric statistics through descriptive statistical analysis and variants. The results of the study show that (1) there are differences in understanding the Biology concept of students at different academic levels (p <0.05), namely understanding concepts at high academic levels is better than at low academic levels; (2) all students at the level of high and low academic abilities show a positive response to the use of e-portfolio which includes three aspects, namely (a) the benefits of using e-portfolio, (b) completion of tasks, and (c) student attitudes and interests.