Objectives In order to enable college students majoring in animal science to proficiently master the theories and techniques of applying animal biotechnology. Methods Teaching theory introduced methods including flipped classroom, online teaching, and knowledge quiz, while teaching practice adopted activities including connecting scientific projects, integrating industry, academia, and research, and participating in innovative projects for college students in the teaching of the second classroom for Animal Biotechnology. Results Students gained a relatively deep grasp and understanding of the theoretical knowledge of the course, especially with the best effect of understanding the forefront and hot topics of animal biotechnology through exploring the reform of teaching theory in the second classroom. The reform and application of multiple practices enabled students to proficiently apply knowledge learned from the course to design solutions and apply professional skills to solve practical problems. The number of students scored above 90 points was significantly higher than those who did not participate in the reform of teaching in second classroom for animal biotechnology, and there was no student scored below 60 points. Conclusions The reform of teaching in second classroom for Animal Biotechnology have made up for the shortcomings of traditional teaching, which is conducive to students better mastering the professional knowledge and skills of animal biotechnology and improving the effectiveness of teaching.