Effect of gradient spoiling in gradient recalled echo (GRE) based two-dimensional phase contrast magnetic resonance imaging (2D PC-MRI) sequence was studied by theoretical analysis and experimental research. 2D PC-MRI simulation was applied to analyze the effect of gradient spoiling on flowing protons. Gradient spoilings of 0π,2π,4π,6π,8π and 10π were employed for flow phantom and volunteer experiment, and the relative flow rates through calculation of imaging results were obtained. Results showed that there was obvious error between the measured flow and setting values when the gradient spoilings of 0π and 2π were applied in the flow phantom experiment. There were significant errors comparing the group with gradient spoilings of 0π and 2π to other groups’ results in the volunteer experiment. When the gradient spoiling was equal or greater than 4π, the measuring errors between the experiment groups were less than 5%. However, due to the fact that the greater use of gradient spoiling requires more time for imaging, 4π was the optimal gradient spoiling in 2D PC-MRI sequence when considering the image quality and the imaging time.