The importance of bioinformatics in current biological research makes bioinformatics education a required course for pre-service biology teachers to become professional biology teachers. This research aimed to develop e-BIMO as a teaching media for bioinformatics education courses towards STEM literacy, considering that STEM literacy is a part that pre-service biology teachers must possess. We used a Research and Development (R and D) research to develop and validate an educational product, STEM literacy-oriented e-BIMO. We use the ADDIE model to develop digital media, which consists of analysis, design, development, implementation, and evaluation stages. We found that the developed e-BIMO model is highly feasible to be implemented more widely because it can support bioinformatics education courses. The content in e-BIMO consists of the scope and role of bioinformatics in daily life, exploring various biological databases, applying BLAST analysis on the theme of genetic diseases, carrying out DNA tracking on the theme of forensic science, carrying out Alignment and creating phylogenetic trees on the topic in the theme of evolution. Pre-service biology teachers agree that e-BIMO is very interesting, has excellent ease of access, a language style that is easy to understand and informative, increases reading motivation, and supports independent learning. So, we conclude that the STEM literacy-oriented e-BIMO has been developed successfully and it can used to support learning in bioinformatics education in the 21st century.