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Protein Engineering is a core compulsory course of biotechnology major, which is the first-class undergraduate major being constructed in Shanxi Province. In view of the problems of single teaching mode of Protein Engineering, such as insufficient students' participation, short teaching time, and expensive experiment cost, the course team carried out the reform and practice of teaching mode for this course, and put forward a new teaching strategy. Under the guidance of the "Golden Course" standard for advancement, innovation and challenge, the course team developed the materials for massive open online courses (MOOC), and carried out the online and offline mixed teaching of Protein Engineering based on BOPPPS+flipped classroom by using the Chao-Xing Fan-Ya network teaching platform. Through this, a comprehensive, systematic and dynamic new teaching system of Protein Engineering was developed. Using the teaching mode based on BOPPPS+flipped classroom, the offline classroom teaching was combined with students' online self-study and homework completion, chapter test and discussion, and this mixed teaching mode was fully integrated into the flipped classroom. After three rounds of teaching practice, the course team had developed a complete, reproducible, scientific and reasonable online and offline mixed teaching mode, which included course materials preparation, exploring experiment guidance, classroom discussion design and course performance evaluation. The online and offline mixed teaching mode of Protein Engineering based on BOPPPS+flipped classroom was helpful for students to improve their autonomous learning ability, to be deeply engaged in the whole teaching process, and to develop a comprehensive and profound understanding of Protein Engineering. This teaching mode improved the teaching quality of Protein Engineering, and facilitated students to learn other follow-up professional courses. Moreover, it provides a reference for the course teaching reform.