Objective: To understand high school students’ mastery of first aid knowledge and their needs for first aid training, analyze the influencing factors of their mastery of first aid knowledge, and provide a reference basis for targeted first aid training for high school students. Methods: A total of 930 high school students from three high schools in Nanchong City, Sichuan Province, were selected as the research subjects using the convenience sampling method. A self-made questionnaire was used for the survey. SPSS 25.0 software was employed for data description and analysis. Results: The average score for high school students’ mastery of first aid knowledge was (12.23 ± 4.39), with a pass rate of 29.5%. There were significant differences in the correct response rates for each question (4.62% to 92.80%). 92.9% of high school students expressed willingness to participate in first aid training. Multivariate logistic regression analysis revealed that participation in first aid training (not participating in training: OR = 2.524, 95%CI = 1.858–3.428) was an influencing factor for high school students’ mastery of first aid knowledge. Conclusion: High school students have insufficient mastery of first aid knowledge, and the correct response rates need to be improved. It is essential to enhance the popularization of first aid knowledge among high school students and implement diversified training formats and courses.