Puncturing an arteriovenous fistula (AVF) in chronic hemodialysis (HD) patients is a source of difficulty even for experienced dialysis nurses. The objective of this study was to compare the failure rate of AVF puncture by the usual technique (visual or by palpation) and after ultrasound location. This nonrandomized prospective single-center study was carried out at the Safi HD Hospital Center for 16 weeks in two successive phases, with the time between each phase dedicated to training nurses in locating punctures by ultrasound. We first evaluated 30 HD patients (300 HD sessions, i.e., 600 punctures) without ultrasound location, then the same patients (300 sessions, i.e., 600 punctures) using ultrasound location for all punctures. Ultrasound location carried out by a single nurse did not show a significant reduction in the rate of failure to puncture the AVF compared with the standard method (2.6% vs. 4%, P = 0.07). However, the rate of complications at the puncture site, recourse to medical advice, or mobilization of a second nurse were significantly reduced (P < 0.05). This single-center study showed no advantage of this approach for the effective cannulation of AVF in HD patients known to be not difficult to cannulate.