Objective To develop an evidence-based practice protocol for initial puncture management of Autologous Arteriovenous Fistulas (AVF) in hemodialysis patients based on optimal evidence, and to evaluate its clinical application effects. Methods Using evidence-based nursing methodologies, we systematically retrieved, evaluated, and synthesized the best evidence on initial puncture management for AVF in hemodialysis patients.Guided by a knowledge translation model, we screened the synthesized evidence based on clinical contexts and stakeholder preferences to develop an evidence-based practice protocol, which was applied in clinical practice from October to December 2024. Results Eighteen pieces of best evidence were summarized and categorized into six themes: team formation and personnel training, timing of the first puncture, pre-puncture assessment, puncture methods, pressure after needle withdrawal, management of puncture complications.After the application of the protocol, the success rate of first puncture for new fistulas increased from 75.36% to 92.40%, and the incidence of puncture-related injuries decreased from 44.93% to 15.20% （both P＜0.05）; the scores of nursing staff′s relevant knowledge assessment increased from （13.10±2.32） points to （17.75±1.57） points, and the execution rate of 9 review indicators increased from 0-86.23% to 81.29-100.00% （all P＜0.05）. Conclusion Evidence-based practice for initial puncture management of AVF in hemodialysis patients can standardize nursing staff′s puncture procedures, improve their relevant knowledge, therefore increase the success rate of first punctures during the initial activation of AVF and reduce the occurrence of puncture complications.