In clinic, intima and media thickness are the main indicators for evaluating the development of atherosclerosis. At present, these indicators are measured by professional doctors manually marking the boundaries of the inner and media on B-mode images, which is complicated, time-consuming and affected by many artificial factors. A grayscale threshold method based on Gaussian Mixture Model (GMM) clustering is therefore proposed to detect the intima and media thickness in carotid arteries from B-mode images in this paper. Firstly, the B-mode images are clustered based on the GMM, and the boundary between the intima and media of the vessel wall is then detected by the gray threshold method, and finally the thickness of the two is measured. Compared with the measurement technique using the gray threshold method directly, the clustering of B-mode images of carotid artery solves the problem of gray boundary blurring of inner and middle membrane, thereby improving the stability and detection accuracy of the gray threshold method. In the clinical trials of 120 healthy carotid arteries, means of 4 manual measurements obtained by two experts are used as reference values. Experimental results show that the normalized root mean square errors (NRMSEs) of the estimated intima and media thickness after GMM clustering were 0.104 7 ± 0.076 2 and 0.097 4 ± 0.068 3, respectively. Compared with the results of the direct gray threshold estimation, means of NRMSEs are reduced by 19.6% and 22.4%, respectively, which indicates that the proposed method has higher measurement accuracy. The standard deviations are reduced by 17.0% and 21.7%, respectively, which indicates that the proposed method has better stability. In summary, this method is helpful for early diagnosis and monitoring of vascular diseases, such as atherosclerosis.