Objective To explore the application effect of multi-track teaching mode in ophthalmology clinical probation teaching. Methods A total of 61 undergraduate students majoring in clinical medicine of grade 2018 and grade 2019 who had clinical practice in the Department of Ophthalmology, Beijing Friendship Hospital, Capital Medical University in 2022 and 2023 were selected as the research objects. A total of 26 undergraduates in grade 2018 were set as the control group, and the traditional teaching method was adopted. The 35 undergraduate students in grade 2019 were set up as experimental group, and multi-track teaching mode was adopted. In this study, a combination of formative evaluation and summative evaluation was used to test the teaching effect, comparing the theoretical examination scores, comprehensive ability scores and student satisfaction scores of the two groups of students. Results The theory examination score of experimental group (83.09±7.70) was higher than that of control group (82.69±11.18). The comprehensive ability score of experimental group (85.20±6.94) was higher than that of control group (81.85±9.55), and the difference was statistically significant (P＜0.05). The satisfaction score of the students with the teachers (99.25±1.99) was significantly higher than that of the control group (96.77±2.87), and the difference was statistically significant (P＜0.05). Conclusion In the process of ophthalmology clinical probation teaching, the application of multi-track teaching mode has better effect and higher teaching satisfaction, which is worthy of reference in ophthalmology clinical teaching.