Objective To confirm the role of dipotassium ethylene diamine tetraacetate (EDTA-K₂) as the preferred anticoagulant in platelet counting using automatic hematology analyzer, and to explore the cause of pseudothrombocytopenia (PTCP) induced by EDTA-K₂ and the corrective measures for EDTA-K₂-associated PTCP (EDTA-PTCP). Methods Blood specimens were collected from 25 healthy volunteers admitted to our hospital from Jun. 2015 to Jun. 2016 and were separately anticoagulated with the anticoagulant EDTA-K₂, sodium citrate and heparin sodium; blood specimens were also collected from 10 patients with EDTA-PTCP in our hospital and were separately anticoagulated with EDTA-K₂ or EDTA-K₂+amikacin. Blood platelet in healthy volunteers and EDTA-PTCP patients was tested by automatic hematology analyzer and manual platelet counting. Results In healthy volunteers, there was no significant difference in the platelet counts between automatic hematology analyzer and the manual platelet counting with EDTA-K₂ anticoagulation for 30 min (P>0.05); and a significant difference in the platelet count was noted between automatic hematology analyzer and the manual platelet counting in the blood specimens with sodium citrate or heparin sodium anticoagulation for 5, 15, 30 and 60 min (P<0.01). The platelet count by automatic hematology analyzer was significantly lower than that by manual platelet count in the blood specimens of EDTA-PTCP patients with anticoagulation for 0, 30, 60, 90 min (P<0.01); after adding amikacin, the platelet count increased with the prolongation of time in these patients with EDTA-K₂ anticoagulation, and there was no significant difference between automatic hematology analyzer and manual platelet count at anticoagulation 90 min (P>0.05). Conclusion Anticoagulation effect of EDTA-K₂ is superior to sodium citrate and heparin sodium, and EDTA-K₂ can be used as the preferred anticoagulant in platelet detection using automatic hematology analyzer. Amikacin can correct EDTA-PTCP in the patients with anticoagulation of EDTA-K₂.