To investigate the effect of serum SCUBE-1 and EndoCan levels on arteriovenous fistula (AVF) thrombosis in hemodialysis (MHD) patients. Methods Patients with MHD and AVF who were treated in our hospital from June 2018 to June 2020 were selected and followed up for 24 months. According to whether thrombosis occurred in AVF, they were divided into thrombosis group and non-thrombotic group. We compared the AVF thrombosis between two groups of MHD patients. Univariate and multivariate analysis analyzed the risk factors for AVF thrombosis; Receiver operating characteristic (ROC) curve analysis analyzed the predictive value of serum SCUBE-1 and EndoCan levels alone and in combination to predict AVF thrombosis in MHD patients. Results A total of 232 MHD patients were included. During the 24-month follow-up process, 41 patients developed AVF. The levels of SCUBE1 and Endocan in the thrombosis group and the non-thrombotic group were statistically significant (P＜0.05). Besides, the age, diabetes, hemoglobin, C reactive protein, low density lipoprotein cholesterol, hypotension and unfractionated heparin anticoagulation between two groups were statistically significant (P＜0.05); Multivariate COX regression showed that the history of diabetes, dialysis hypotension, unfractionated heparin anticoagulation, SCUBE1 and Endocan levels were risk factors for thrombosis (P＜0.05); The AUCs of SCUBE1 and Endocan alone and in combination to predict AVF were 0.813, 0.912, and 0.939, respectively. Conclusion Increased levels of SCUBE1 and Endocan are risk factors for AVF thrombosis; SCUBE1 and Endocan have high diagnostic value for the prediction of AVF thrombosis, and AVF patients with serum EndoCan＞0.98ng/mL and SCUBE-1＞40ng/mL should be vigilant its risk of thrombosis