Background <p>Work-related biological exposure continues to pose significant health risks for nursing students due to their limited clinical skills and experience. Web-based learning provides standardized, flexible training and peer teaching enhances engagement through relatable knowledge exchange, and the combined potential of these two methods in work-related biological exposure education has yet to be fully explored. This study aimed to investigate the effect of web-based learning with peer-facilitated discussion on work-related biological exposure education among nursing students.</p> Methods <p>This quasi-experimental study was conducted at a tertiary teaching hospital in southwest China from June 2022 to December 2023. The nursing students who began their internship in June 2022 were defined as the control group and were trained using traditional lectures, while those who started their internship in June 2023 were designated as the experimental group and were instructed through web-based learning with peer-facilitated discussion. After the training on work-related biological exposure for nurses in hospital settings, a test was immediately conducted to assess the participants’ knowledge about work-related biological exposure. Six months after each group completed the training, participants were surveyed about their behaviors and experiences related to work-related biological exposure risk. A comparison of knowledge and practices regarding work-related biological exposure risk was conducted between the two groups.</p> Results <p>There were no statistically significant differences between the two groups in participants’ gender, age, and educational status (<i>p &gt;</i> 0.05). The total scores of knowledge about work-related biological exposure in the experimental group were higher than those in the control group after training (18.80 ± 1.77 vs. 14.90 ± 2.11, <i>p &lt;</i> 0.05). At the 6-month follow-up survey, the experimental group reported an increase in safe behaviors and a decrease in risky behaviors related to work-related biological exposure compared to the control group (<i>p</i> &lt; 0.05). There was a difference in the rate of nursing students who reported an experience of work-related biological exposure between the two groups, with the control group reporting a higher rate of exposures and lower compliance with reporting requirements than the experimental group (<i>p &lt;</i> 0.05).</p> Conclusion <p>The study demonstrated that web-based learning with peer-facilitated discussion may improve the knowledge and behaviors related to work-related biological exposure. These findings highlight the potential of this effective teaching method in reducing the risk of work-related biological exposure among nursing students, with potential applicability to other healthcare programs.</p>