Cryopreservation of red blood cells (RBCs) is fundamentally important to modern transfusion medicine. Currently, organic solvent glycerol is utilized as the state-of-the-art cryoprotectant (CPA) for RBC cryopreservation. However, glycerol must be removed before RBC transfusion to avoid intravascular hemolysis via a time-consuming deglycerolization process with specialized equipment (e.g., ACP 215), thus limiting the clinical use of frozen RBCs. Herein, we report novel biocompatible CPA formulations combining betaine with membrane stabilizers (disaccharides or amino acids), which can achieve outstanding efficiency for RBC cryopreservation directly using whole blood without any separation process. Most importantly, because of the osmotic regulation capacity of betaine, a simple and fast one-step method can be used for CPA removal, which is significantly superior to the current multistep deglycerolization process. This work offers a promising solution for highly efficient and solvent-free RBC cryopreservation and holds great potential for improving the long-term storage and long-distance distribution of RBCs.