〔Abstract〕 Objective To investigate and analyze the effect of different preparation times on the quality of leukocyte-reduced red blood cell suspension. Methods A total of 160 blood donation samples from the same batch collected on May 21, 2023 in Nanping Central Blood Station were randomly divided into a study group and a control group, with 80 samples in each group. The control group was stored in a refrigerator at 4 °C after blood collection. When the blood temperature dropped to about 10 °C, white blood cells were filtered and centrifuged to prepare leukocyte-reduced red blood cell suspension and plasma. The study group stored the blood in a refrigerator at 4 °C after blood collection. When the blood temperature dropped to about 10 °C, white blood cells were filtered, but not centrifuged. According to different storage time, the filtered blood was divided into four groups: study group 1 (20 samples, stored for 1 day), study group 2 (20 samples, stored for 5 days), study group 3 (20 samples, stored for 9 days), study group 4 (20 samples, stored for 14 days) were removed at the end of storage, and the leukocyte-reduced red blood cell suspension and plasma were centrifuged. The differences of hemolysis rate, plasma free hemoglobin concentration, blood routine index and hemorheology index were compared at the end of blood storage. Results There was no significant difference in red blood cell hemolysis rate between the study group and the control group (P > 0.05). The plasma free hemoglobin concentration in the study group was significantly higher than that in the control group, and with the extension of preparation time, the plasma free hemoglobin concentration was also significantly increased, and the differences were statistically significant (P < 0.001). There were no significant differences in the residual amount of white blood cells, the volume of white removed suspension red blood cells and hematocrit among different groups (P > 0.05). The erythrocyte aggregation index, erythrocyte rigidity index and erythrocyte electrophoresis index of the study group were significantly higher than those of the control group (P < 0.001), and with the extension of preparation time, erythrocyte aggregation index, erythrocyte rigidity index and erythrocyte electrophoresis index were also significantly increased, and there were significant differences between groups (P < 0.001). There was no significant difference in the erythrocyte degeneration index comparison among the different groups (P > 0.05). Conclusion The extension of preparation time of leukocyte-reduced red blood cell suspension can lead to the increase of free hemoglobin concentration, resulting in the increase of erythrocyte aggregation index, erythrocyte rigidity index and erythrocyte electrophoresis index, so the preparation time of leukocyte-reduced red blood cell suspension should be strictly controlled.