Background: The blood vessel in the upper extremity arteriovenous fistula (AVF) is deep in the tissue, and cannulation in AVF is frequently associated with blood oozing, hematoma, or aneurysm. This study evaluated the performance of color ultrasound in-plane guided cannulation technique during upper extremity high-AVF cannulation in patients with hemodialysis. Methods: A total of 40 patients with hemodialysis who needed cannulation in upper extremity AVF were recruited in the study, and the patients were randomly divided into observation group and control group. Color Doppler ultrasound was used to guide cannulation in the observation group and in the control group blind cannulation method was applied. The success rate of onetime cannulation, the incidence of subcutaneous hematoma, oozing, and pain caused by incorrect fistula cannulation as well as the satisfaction score of the patients were compared to evaluate the effect and advantages of color ultrasound-guided cannulation. Results: The one-time success rate of internal fistula cannulation in the observation group (98.71%) was significantly higher than that in the control group (88.27%). The incidence rates of hematoma, oozing, pain, and total failure events were significantly reduced in the observation group. The average satisfaction degree in the observation group was also significantly higher than that of the control group. Conclusion: Ultrasonic-guided cannulation effectively enhances the success rate of cannulation in upper extremity AVF, reduces the incidence of cannulation failures, and improves the satisfaction level in the patients.