Molecular Biology Experiments is a core foundational course for biology majors, playing a crucial role in cultivating students’ practical abilities and research literacy. In response to the problems of abstract and difficult-to-understand principles, poor demonstration effects, low student participation, and single evaluation methods in traditional teaching, this study is based on the actual situation of local colleges and universities, and carries out systematic teaching reform practices based on the teaching conditions of our school. By building a “dual teacher” teaching team, optimizing the three-level experimental project system of “basic comprehensive expansion,” constructing a hybrid teaching mode based on self-built online courses and virtual simulation platforms, and implementing a diversified assessment and evaluation mechanism throughout the entire teaching process, students’ learning initiative has been effectively stimulated, and their self-learning ability, experimental operation level, and comprehensive problem-solving ability have been significantly improved. This reform provides a replicable and operable practical path for local universities with similar teaching resources to carry out molecular biology experimental teaching.