Objective To design and verify an analytical method for the cross contamination of Beckman AU5811 automatic biochemical analyzer's reagent needle and stir bar and find the reasons, and make effective measures to avoid contamination. Methods Use the inner ring items (ALT,TBIL,GGT,ALB, TBA,LDH1,CK,Cr,GLU,CO2,TC,HDL-C,APOA1,Ca,Fe,Cu,HCY,AFU,PA) as theexperiment objects and do the cross contamination experiment according to the test order. The standard for the cross contamination level is limited to 95-105% among all items. Results TC and HCL-C were found to have contaminated TBA both in reagent needle and stir bar model, Ca contaminated Cr in the stir bar model; Cu contaminated Fe in the reagent needle model; GGT,ALB and LDH1 contaminated CO2 in stir bar model; TC and TBIL contaminated Cu in the stir bar model. Conclusion The method used in this experiment can effectively identify and analyze the cross contamination of all the items in Beckman AU5811 automatic biochemical analyzer. In addition, it can help to adopt corresponding measures to reduce cross contamination.