The present application relates to a fixation treatment method for an in-vitro breast cancer axillary lymph node tissue specimen, and belongs to the technical field of sample preparation for testing.A to-be-tested in-vitro breast cancer axillary lymph node dissection tissue specimen is immersed in a stationary liquid for 2 to 24 hours. After washing, the specimen is immersed in a hydrochloric acid solution for 30 to 120 minutes, then rinsed, and soaked in an alkali solution for 30 to 120 minutes. A digestive solution contains 0.1-2 mg/ml of protease K, 1-100 mg/ml of trypsin and 0.1-2 mg/ml of EDTA, and after constant temperature water bath treatment for 1-10 hours, lymph nodes are cut, routinely dehydrated, embedded, sliced and stained to complete the treatment. The fixation treatment method for the in-vitro breast cancer axillary lymph node tissue specimen can not only soften and dissolve adipose tissue effectively, and ensures timely and sufficient fixation of the lymph nodes, andthe number of detected lymph nodes and accurate lymph node metastasis positive rate are effectively improved.