With the aging population and lifestyle changes, the number of hemodialysis (HD) patients increases year by year. The arteriovenous fistula (AVF) is the gold standard vascular access used to access the blood for HD treatment. Since the status of stenosis affects HD efficiency, current clinical practices usually use a Doppler ultrasound imaging system to assess the parameters of the stenosis, such as the degree of stenosis (DOS). Unfortunately, this is a very time-consuming task. Furthermore, it is difficult to stably maintain the ultrasound probe for a prolonged period to give doctors clearer or reproducible images. In this study, a robotic ultrasound system (RUS) with ultrasound sequential imaging analysis was designed to evaluate the DOS of the AVF. The sequential imaging analysis was capable of image smoothing and vessel boundary detection. It enabled clinicians to mark the thickness of the plaque for further processing. Finally, the system was used to reconstruct 3D models of fistulas and calculated the DOS for clinical assessment. We also designed a pressure sensing module attached to the ultrasound probe to prevent the probe from coming loose, vibrating, and exerting abnormal pressure on the skin. In the phantom test, the results showed that the error of the DOS that was calculated by RUS was less than 3%. The results of clinical trials obtained from ten patients show that the error between the RUS and clinicians’ measurement was about 10% and had a highly linear correlation (R Square > 0.95). In addition, the reproducibility error was about 3% and could effectively save about 46% of the time during clinical examinations.