The clinical transfusion of red blood cells (RBCs) has provided the greatest number of cryobiology applications in the case of rare blood groups and antibody problems, as well as civil and military disasters. The main technical difficulty with the current clinical technique is the removal of high concentration glycerol (20% or 40%) after thawing. Reducing the probability of intracellular ice formation (IIF) as well as preventing the solution effect are crucial to ensure RBCs avoid cryoinjury. Here, the non-permeating cryoprotectant trehalose was used to dehydrate RBCs before freezing. Furthermore, with the substitution of the low concentration glycerol (5% or 7.5%) for the intracellular remaining water, the bulk of RBCs were successfully cryopreserved to obtain a nearly 95% high survival rate with rapid cooling via EP tubes. Additionally, the washed RBCs after cryopreservation maintained their morphology, deformability, ATP, and 2-3 DPG levels, and all of them met the clinical standards for transfusion safety. Moreover, the whole addition and washing process was simple and easy to operate and could be completed within 30 min, which is crucial for emergency uses. This method will provide more potential for current clinical RBCs cryopreservation practices.