Objective: To conduct a pre-course survey on the graduate students who participated in the Molecular Medicine Laboratory Skills Training Course in the Zhongshan Ophthalmic Center of Sun Yat-sen University to provide a reference for the preparation of the course. Methods: We asked the doctoral and master’s students who participated in the training courses of molecular medical experimental skills in 2020 and 2021 to take a pre-course questionnaire survey and analyzed the results by the students’ pre-course command of theoretical knowledge and pre-training operational experience in 15 lessons of special topics or techniques. Results: A total of 155 students completed the pre-course questionnaire survey. Our results showed that there were 14 lessons in which more than 25% of the students were not familiar with the theoretical knowledge, whereas there were 6 lessons in which less than 10% of the students had high operational proficiency. The students majoring in the molecular medicine had better prior knowledge of the techniques: there were only 9 lessons in which more than 25% of the students were not familiar with the theoretical knowledge, whereas there were 12 lessons in which less than 10% of the students had high operational proficiency. The four lessons with worst prior knowledge were cell proliferation, flow cytometry, image acquisition, and omics analyses. We grouped the 15 lessons into 7 categories: the ophthalmic experimental data collection, the omics technology, the data analyses, the cell technology, the protein technology, the tissue analyses, and the molecular technology, presenting by the level of the students’ pre-training proficiency from low to high. Conclusion: The first-year graduate students were very weak in basic knowledge and had little experience in techniques before joining the molecular medicine training courses. It is urgent to improve the laboratory skills training system and strengthen the construction of the curriculum system.