Abstract Raman tweezers have been used to study single blood cells in vivo without requiring invasive procedures. In the present study, we use optical tweezers to capture single blood cells (RBCs) in the microvessel of a mouse ear. Without the use of any invasive procedures, we were able to obtain the Raman spectra of single red blood cells from a mouse ear. An analysis of these spectra indicated that RBCs in arterioles are oxygenated, while those in the capillaries of venules are deoxygenated, those in vitro are similar to that in venules. In addition, hemoglobin in vivo was observed to be more concentrated than that in vitro. By studying the change of the band at 1604cm−1, we concluded that the pH in arterioles is higher than that in venules. The information gained from the single RBCs in vivo is important for the understanding of the reaction of RBCs to changes in their environment and may have many applications in the diagnosis and treatment of red blood cell disorders.