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Introduction: Hemolysis is a common pre‐analytical factor that can influence test results. Here, we explored the influence of hemolysis on nucleated red blood cells (NRBCs) count and tried to illustrate the mechanisms underlying this interference. Methods: From July 2019 to June 2021, 20 preanalytical hemolytic peripheral blood (PB) samples from inpatient at Tianjin Huanhu Hospital were evaluated using Sysmex XE‐5000 automated hematology analyzer. When NRBC enumeration was positive and a flag was triggered, a 200‐cell differential count was performed by experienced technologists on microscopic review. When the manual count was inconsistent with automated enumeration, samples will be re‐collected. Plasma exchange test was performed to verify the influence factors of hemolyzed samples and the mechanical hemolysis experiment mimicking hemolysis that might occur during blood collection was performed to illustrate the underlying mechanisms. Results: Hemolysis led to false‐positive NRBC count and the value of NRBC was positively correlated with the degree of hemolysis. Hemolysis specimen shared a common scatter diagram: a "beard" on WBC/ basophil (BASO) channel and a "blue scatter line" on immature myeloid information (IMI) channel. Lipid droplets were found above the hemolysis specimen after centrifugation. Plasma exchange experiment confirmed that these lipid droplets interfered with NRBCs count. Mechanical hemolysis experiment implied further that broken red blood cells (RBCs) released lipid droplets causing the false‐positive NRBCs count. Conclusion: In the present study, we firstly found that hemolysis could lead to false‐positive NRBCs enumeration, which was associated with lipid droplets released from broken RBCs during hemolysis. [ABSTRACT FROM AUTHOR]