Abstract This paper introduced the application of "four in one" teaching mode of learning, teaching, operating and evaluating in the teaching of organic chemistry experiment taking the synthesis of butyl benzoate as an example. The students' learning enthusiasm was promoted through the way of guiding learning, assisting learning, promoting learning and teaching after learning. Problem-based learning was adopted, and the teacher-student relationship with teacher as the leading and the student as the main body was established, which could motivate students' subjective initiative. Open experiments were selected instead of traditional experiments to train students' experimental skills and scientific literacy. And experimental results were evaluated by three-dimensional evaluation system to reflect the objectivity and authenticity of the evaluation. The practice showed that the "four in one" teaching mode could fully arouse the students' subjective initiative, cultivate the students' experimental skills and interests, and effectively improve the quality of experiment teaching.