Cryopreservation of red blood cells (RBCs) provides great potential benefits for providing transfusion timely in emergencies. High concentrations of glycerol (20% or 40%) are used for RBC cryopreservation in current clinical practice, which results in cytotoxicity and osmotic injuries that must be carefully controlled. However, existing studies on the low-glycerol cryopreservation of RBCs still suffer from the bottleneck of low hematocrit levels, which require relatively large storage space and an extra concentration process before transfusion, making it inconvenient (time-consuming, and also may cause injury and sample lose) for clinical applications. To this end, we develop a novel method for the glycerol-free cryopreservation of human RBCs with a high final hematocrit by using trehalose as the sole cryoprotectant to dehydrate RBCs and using core-shell alginate hydrogel microfibers to enhance heat transfer during cryopreservation. Different from previous studies, we achieve the cryopreservation of human RBCs at high hematocrit (> 40%) with high recovery (up to 95%). Additionally, the washed RBCs post-cryopreserved are proved to maintain their morphology, mechanics, and functional properties. This may provide a nontoxic, high-efficiency, and glycerol-free approach for RBC cryopreservation, along with potential clinical transfusion benefits.