Objective To improve the surgical nursing cooperation ability of orthopedic subspecialty rotating nurses in operating room. Methods A standardized training system for orthopedic subspecialty in operating room was established based on enabling evaluation, including handing strategies of external instruments, which was then used for the training of 36 rotating nurses in orthopedic subspecialty in operating room. Results After the training, the comprehensive assessment results and core competency scores of the 36 nurses were significantly better than those before the training (both P<0.05). Conclusion Establishment and application of the orthopedic subspecialty nursing training system in operating room based on enabling evaluation, effectively improves the surgical nursing cooperation level and handling ability of external instruments of orthopedic subspecialty rotating nurses.