Pre-service biology teachers' environmental literacy is still generally low. It is necessary to improve their environmental literacy through the innovation of open inquiry learning kits. There are two primary aims of this study: (1) to develop appropriate open inquiry learning kits that can foster pre-service biology teachers' environmental literacy in environmental education courses, (2) to examine the effectiveness of open inquiry learning kits in improving environmental literacy. This study used the procedure of the 4-D Thiagarajan model, i.e., four stages: (1) define; (2) design; (3) develop; and (4) disseminate. The products produced are open inquiry lesson plans, student worksheets, and environmental literacy test instruments. The product validation was carried out by experts in the field of environmental education and biology teaching and learning. The preliminary field testing and the main field testing were carried out in environmental education courses. The data were collected through observation, questionnaires, and written tests. The data were analyzed in a quantitative descriptive way through the calculation of the gain score. The results obtained from the research and development are open inquiry learning kits to improve the environmental literacy of students, which as a whole are suitable to use in environmental education lectures based on expert judgment. These open inquiry learning kits are not very effective in improving students' environmental literacy based on the calculations of the gain score due to various shortcomings.