End-stage renal disease (ESRD) is the final stage of chronic kidney disease (CKD). We aimed to analyze the expression differences of serum thrombomodulin (TM), platelet-activating factor (PAF), and P-selectin (CD62P) in patients with autologous arteriovenous fistula (AVF) and the correlation with vascular access function. The case data were retrospectively analyzed. Moreover, 160 patients with AVF maintenance hemodialysis were selected as the AVF group, and 150 healthy participants were selected as the healthy control group. According to the function of vascular access, patients in the AVF group were divided into Group A (n = 50, after the first establishment of AVF), Group B (n = 64, normal vascular access function after hemodialysis treatment), and Group C (n = 46, vascular access failure). Pearson analysis was conducted to explore the correlation between serum TM, PAF, CD62P content, and vascular pathological examination indicators, to evaluate the value of TM, PAF, and CD62P levels in predicting vascular access failure in patients with AVF. The serum levels of TM, PAF, and CD62P were positively correlated with the expressions of CD68 and MCP-1, respectively (p < .001). Serum TM was positively correlated with the levels of PAF and CD62P (p < .001), and PAF was positively correlated with the levels of CD62P (p < .001), respectively. Serum levels of TM, PAF and CD62P were risk factors for vascular access failure in AVF patients (p < .05). The area under the curve of serum TM, PAF and CD62P levels in predicting vascular access failure in AVF patients was 0.879. The serum levels of TM, PAF, and CD62P in AVF patients were correlated with the vascular access function of AVF patients, which was very important for maintaining the stability of vascular access function, and had certain value in predicting vascular access failure/disorder in AVF patients, and could be popularized and applied.