Background <p>The cannulation technique employed by nurses significantly impacts arteriovenous fistula (AVF) patency rates. To ensure optimal outcomes, nurses must strictly adhere to evidence-based protocols and maintain up-to-date knowledge of best practices in cannulation methods.</p> Methods <p>A sequential mixed-methods study was conducted. In May 2024, convenience-sampled nurses from haemodialysis units in Jiangsu Provence, China, completed an evidence-based questionnaire on AVF cannulation practices and associated factors. Subsequently, purposively sampled haemodialysis nurses and nurse managers participated in semi-structured interviews. Quantitative data were analysed using descriptive statistics, and qualitative data were analysed thematically.</p> Results <p>A total of 489 nurses completed the survey, and 10 nurses participated in semi-structured interviews. Quantitative findings revealed that while 89.16% of departments had cannulation plans, only 65.44% had corresponding diagrams and just 62.78% of these included both vessel direction and cannulation sites. Regarding cannulation techniques (multiple selections allowed), although rope ladder puncture (RL) was the most commonly used technique (96.93%), nearly half of the respondents still employed area puncture (AP) (44.38%), despite guideline recommendations against it. Multivariate regression analysis showed that the availability of ultrasound-guided cannulation was negatively associated with the use of AP or buttonhole puncture (BH) (<i>P</i> &lt; 0.05). Higher evidence-based practice (EBP) competency scores (<i>P</i> = 0.006) and years of dialysis experience (<i>P</i> = 0.001) were positively associated with the use of BH. In contrast, the role of head nurse (<i>P</i> = 0.001), the establishment of a vascular access team (<i>P</i> = 0.03) were all negatively associated with the use of BH. Qualitative analysis revealed three key themes: (1) Insufficient implementation of cannulation plans in clinical practice, (2) lack of standardized competency assessment for nurses, and (3) weak EBP awareness and implementation capacity. These findings suggest that the persistent use of suboptimal techniques stems not from a lack of guidelines, but from systemic issues including skill mismatches, insufficient monitoring, and limited EBP integration.</p> Conclusions <p>This mixed-methods study reveals a critical gap between evidence-based guidelines and the clinical reality of vascular access cannulation in hemodialysis settings. While quantitative findings indicate suboptimal adherence to recommended techniques, qualitative insights elucidate that this is not merely a knowledge deficit but is deeply rooted in fragmented responsibility, inadequate competency assessment systems, and weak organizational support for EBP. Therefore, improving patient safety and cannulation outcomes requires moving beyond simple guideline dissemination to implement multi-level interventions. We recommend establishing structured skill validation programs and specialized vascular access teams to foster a culture of accountability and ensure the translation of best evidence into routine clinical practice.</p>