Objective To explore the suitable blood storage time of preoperative autologous blood donation (PABD) so as to provide theoretical basis for ensuring the safety and effectiveness of autologous blood application. Methods Ten patients undergoing orthopedic surgery with PABD indications were selected, and the total blood volume was calculated according to the patient's weight. The autologous blood accounting for 10% of the total blood volume was collected one week before operation. The blood routine and hemodynamics were detected before and after blood collection, and 50 ml autologous blood was reserved to prepare red blood cell (RBC) suspension for research use. The effective oxygen‑carrying amount (Q), oxygen affinity (P50), adenosine triphosphate (ATP), 2, 3‑diphosphoglycerate (2, 3‑DPG), Na+‑K+‑ATPase, Ca2+‑Mg2+‑ATPase, reactive oxygen species (ROS) of the stored autologous blood samples were measured on days 1, 7, 14, 21 and 28 and their correlations were analyzed. Results Compared with those before blood collection, the RBC count, hemoglobin (Hb), hematocrit (Hct), RBC aggregation index and RBC rigidity index decreased after blood collection (P<0.05). Compared with storage for one day, RBC Q value, Na+‑K+‑ATPase activity and Ca2+‑Mg2+‑ATPase activity decreased on days 7, 14, 21 and 28, ATP and 2,3‑DPG content decreased on days 14, 21 and 28, P50 value decreased on days 21 and 28, and ROS content increased on days 7, 14, 21 and 28 (P<0.05). Compared with storage for seven days, RBC Q value, 2,3‑DPG content and Na+‑K+‑ATPase activity decreased at days 14, 21 and 28, while P50 value, ATP and Ca2+‑Mg2+‑ATPase activity decreased on days 21 and 28, and ROS content increased on days 14, 21 and 28 (P<0.05). The ROS content of RBC was positively correlated with storage time (P<0.05); RBC Q value, P50, ATP, 2,3‑DPG, Na+‑K+‑ATPase and Ca2+‑Mg2+‑ATPase contents were negatively correlated with storage time and RBC ROS content (P<0.05). Conclusions PABD RBC can basically maintain normal oxygen‑carrying capacity within 14 days, and the oxygen‑carrying capacity of RBC decreases with the prolonging of storage time, which may be related to the oxidative stress injury of RBC.