Abstract: The profile of red blood cells is an important index for the prevention and diagnosis of various diseases in the medical field. A simultaneous phase-shifting microscopic interference method was proposed to realize the dynamic measurement of the profile of red blood cells. A transmission-type microscopic interference imaging system was set up to measure the red blood cells of New Zealand rabbit with a diameter of 7 μm~8 μm, and a maximum height of 2 μm in a simulated microvessel with an inner diameter of 100 μm. Aiming at the microvesse environment in which the red blood cells were located, a method of extracting the profile of red blood cells based on the phase subtraction of microvessels and a correction method of imaging magnification were proposed. The average diameter and maximum height of the red blood cells in the simulated microvessels are obtained by experiments, which are 7.757 μm and 2.022 μm, respectively. The potential of this method in vivo measurement of RBC profile has been verified.