Successful human red blood cell cryopreservation techniques have been gradually developed in recent decades, with great potential for use in clinical medicine and basic research. The mechanical properties of a single cell are important clues to reveal the physiological and pathological state of the red blood cell, but they have not been used to assess the physiological state of the cell after cryopreservation. Herein, we investigated the effects of cryopreservation processes on human red blood cell biomechanics by means of a microfluidic, label-free, synchronous, and nondestructive electrodeformation assay. We found that the effects of viscoelasticity of the red blood cell membrane between permeable and impermeable cryoprotectants were different. Our findings showed that high freeze-thaw recovery did not mean that the recovered cells had excellent viscoelasticity. The results demonstrate that single-cell viscoelasticity is an irreplaceable indicator for assessing the quality of the recovered cells and help us to deepen understanding of the cryoinjury mechanism. Assessment of the single cell viscoelasticity offers significant potential for application in optimizing the cryopreservation process and screening optimal cryoprotectants.