【Abstract】Objective To explore the feasibility of handheld ultrasound examination for real- time guidance on cannulation of new arteriovenous fistula (AVF), and to compare its effects to the standard cannulation method. Methods This was a single center, randomized and prospective study comparing the handheld ultrasound examination-guided cannulation to new AVF with the standard cannulation method. Sixty end stage renal disease patients with a new and mature wrist radial-cephalic AVF treated in this hemodialysis center were enrolled in this study. Patients with new AVF received either standard cannulation (control group) or ultrasound guided cannulation (study group) for one week. Cannulation manipulation and complications were observed. Results Baseline data including age, body weight index, gender and comorbidities had no statistical significances between the two groups (P＞0.05). Compared to control group, study group yielded longer assessment time before cannulation (123.2±47.7s vs. 40.4±30.6s;p t=19.145, P＜0.001), longer cannulation time (48.6±20.2s vs. 32.0±30.7s; t=2.556, P=0.011), higher success rate (100.0% vs. 96.5%; χ2=6.393, P=0.011), and lower number of cannulation attempts (1.0 vs. 1.0±0.2; t=6.519, P＜0.001). The puncture injury rate was totally 5.2% in the patients; cannulation injury rate was lower in study group than in control group (2.8% vs. 7.3%; χ2=4.150, P=0.042). Change to catheterization for blood access due to cannulation injury was lower in study group than in control group but without statistical significance (1.1% vs. 5.6%; χ2=2.759, P=0.097). No puncture into arteries and infection due to cannulation were found in both groups. Conclusion Handheld ultrasound examination for real-time guidance on cannulation for new AVF can increase the success rate and decrease the cannulation-related complications. It is a safe and useful method for blood access to be used widely in clinical practice.