Objective To evaluate the application effect of a Web-based three-dimensional (3D) interactive system for arteriovenous fistula puncture training in medical education. Methods A total of 120 novices without vascular puncture experience from 4 medical colleges between September 2023 and June 2024 were randomly divided into control and trial groups, with 60 novices in each group. The control group received conventional training with silicone vascular models and peer simulation, while the trial group used the Web-based 3D interactive teaching system. Training time, puncture accuracy, and satisfaction scores were compared between the two groups. Results The differences in gender, age, student level, and source institutions between the trial group and the control group were not statistically significant (P>0.05). Compared with the control group, the trial group had shorter training time [(45.0±8.0) vs. (75.0±12.0) min; t=–16.200, P<0.001], higher puncture accuracy [(90.2±5.1)% vs. (66.8±8.4)%; t=18.570, P<0.001], and higher satisfaction (4.6±0.4 vs. 3.2±0.6; t=17.800, P<0.001). Conclusion A Web-based 3D interactive arteriovenous fistula puncture system can significantly shorten training time, improve operational accuracy, and enhance learning satisfaction, providing an efficient and standardized model for medical skill training.