Pre-service science teachers need to be aware of eco-literacy and biotechnology as they will teach science subjects to students from the perspectives of 21st-century skills. This study aimed to develop a web-based module on biotechnology and examine preservice science teachers’ eco-literacy skills after using the developed module. The study used a research and development design adopting Molenda’s product development model (Analysis, Design, Development, Implementation, and Evaluation/ADDIE). There were 60 students majoring in science education as the research participants. The data were collected using a needs analysis questionnaire, a curriculum analysis form, a validation sheet, tests, and a response questionnaire. The obtained quantitative data were analyzed using IBM SPSS 25 with descriptive statistical tests with Swanson’s quantifications. Results showed that the web-based module on biotechnology could be developed through ADDIE stages with experts’ high validity results (M = 4.59, valid with revision). The module significantly improved the teachers’ eco-literacy skills on biotechnology topics (F = 14.053, p = .000, N = 60). This result implies that the web-based module on biotechnology can assist preservice science teachers in improving their eco-literacy skills.