This study aims to determine the effectiveness of the STEAM (Science, Technology, Engineering, Arts, and Mathematics)-based biotechnology module equipped with flash animation for learning biology in high school. This research was conducted in a high school, involving 32 students of Year XII. The research design was the design model for development of R2D2 with one group pretest-posttest design as effectiveness tests. This study utilized a learning design, biotechnology modules equipped with flash animation, instrument testing, validation sheet and questionnaire as research instruments. Data validation and the questionnaire results were then converted into qualitative data into a category. The effectiveness test data were analyzed using the N gain. The results of the validation material, media and users indicated that the module was valid with the final score of 89.85. Students' responses to the module in terms of readability, clarity of presentation, the attractiveness and usefulness of the results were in very good category (81.2%). The effectiveness test showed that result of post-test greater than pre-test and the value of N gain were in the high category (0.72).