The cycle of Hemoglobin oxygenation and deoxidation plays an important role in driving structure and regulating function of red blood cell in vivo, has attracted wide attention. But it has not yet been reported about any studies on the oxygen-carrying function of individual living RBC in vitro with the incubation time. This study involved that using confocal Raman scanning microscopic technique, collecting the Raman spectra of living erythrocyte cultured in vitro at different time, analysing the peaks (1 636, 1 562 cm-1) with characterization of hemoglobin oxygen carrying capacity and the amide Ⅲ band (1 240～1 300 cm-1) with sensitivity to conformation for understanding those changes both of hemoglobin oxygen carrying capacity and protein conformation over time. Meanwhile, its corresponding surface micromorphology was observed via scanning electron microscopy (SEM). The results indicated that the during 24 h hemoglobin with stable structure and normal allosteric collaborative function occurred alternately with the oxygen uptake of increase and decrease and conformation R state and T state, while double concave disk red blood cells observed under SEM also alternately between stretch and shrink. The conclusion not only provides a multi-level characteristic parameter from a single living cell for the study of RBC oxygen-carrying function in vitro, but also the potential research ideas for the screening of the active component, the evaluation of drug efficacy and toxicity for RBC in vitro.