Objective: To explore the application effect of typical critical situation simulation teaching in the operating room in the stratified training of nurses, and to provide practical basis for optimizing the training system of operating room nurses and improving their clinical emergency response capacity. Methods: A total of 50 operating room nurses in a tertiary hospital were selected and divided into N0~N1 grade (junior group), N2 grade (middle group) and N3–N4 grade (senior group) according to their professional titles, working years and post capabilities. A 6-month stratified training with typical critical situation simulation was implemented. Before and after the training, theoretical assessment, operational skill scoring, emergency response capacity scale and professional identity scale were used to evaluate the effect, and training satisfaction was collected simultaneously. Results: After training, the scores of theoretical knowledge, operational skills and emergency response capacity of nurses in the three groups were significantly higher than those before training (p < 0.05), and the improvement range of team cooperation capacity in the senior group was better than that in the junior group (p < 0.05); the score of professional identity increased significantly (p < 0.05), and the total training satisfaction reached 95.0%. Conclusion: The stratified teaching with typical critical situation simulation in the operating room can effectively improve the professional competence and professional identity of nurses at all levels, adapt to the needs of standardized training, and is worthy of popularization and application.