Ki-67 standard reference card (SRC) and artificial intelligence (AI) software were used to evaluate breast cancer Ki-67LI. We established training and validation sets and studied the repeatability inter-observers.A total of 300 invasive breast cancer specimens were randomly divided into training and validation sets, with each set including 150 cases. Breast cancer Ki-67 standard reference card ranging from 5 to 90% were created. The training set was interpreted by nine pathologists of different ages through microscopic visual assessment (VA), SRC, microscopic manual counting (MC), and AI. The validation set was interpreted by three randomly selected pathologists using SRC and AI. The intra-group correlation coefficient (ICC) were used for consistency analysis.In the homogeneous and heterogeneous groups of validation sets, the consistency among the pathologists that used SRC and AI was very good, with an ICC of>0.905. In the validation set, using SRC and AI, three pathologists obtained results that were very consistent with the gold standard, having an ICC above 0.95, and the inter-observer agreement was also very good, with an ICC of>0.9.AI has satisfactory inter-observer repeatability, and the true value was closer to the gold standard, which is the preferred method for Ki-67LI reproducibility; While AI software has not been popularized, SRC may be interpreted as breast cancer Ki-67LI's standard candidate method.© 2022. The Author(s).