Background <p>Recognizing deteriorated vital signs and handling acute illness appropriately is crucial for hospitalized patients. However, healthcare professionals often face criticism for inadequate first aid skills. We aim to evaluate the teaching effect of simulation-based stratified first aid skills training on physicians.</p> Methods <p>A cohort study was conducted with 263 physicians as participants. They were stratified into three levels (groups) according to the clinical working time and different disciplinary backgrounds: basic first aid skills training (63 physicians), senior first aid skills training (159 physicians), and advanced case scenario simulation training (41 physicians). The teaching effect on objective skills test scores, pre-post training self-estimate scores and confidence, and training satisfaction was evaluated.</p> Results <p>Based on the objective evaluation criteria, the average skills test scores of three different levels of skills training groups were higher than 90, and the highest average skills test score was the basic first aid skills training group. In three different levels of skills training groups, the self-estimate scores within the range of 100–90 and 89–80 after the training were statistically significantly higher than before the training (<i>P</i> &lt; 0.01). The confidence in managing clinical emergencies after the training was significantly higher than before the training (<i>P</i> &lt; 0.01).</p> Conclusion <p>The simulation-based first aid skills training stratified into three levels is effective in helping high-level first aid proficiency in physicians. It provides a stratified training pattern according to the clinical working time and different disciplinary backgrounds of physicians.</p>