Objective This prospective and self-controlled study was designed to detect whether serum folate and vitamin B12 concentration improved in hemodialysis patients treated with high flux dialysis. Methods A total of 30 patients on maintenance low flux hemodialysis were included. After switching to high flux dialysis, dialysis prescription was unchanged except the change of dialyzer. Ultrapure dialysate was implied. Pre-dialysis blood samples were collected for measuring serum folate and vitamin B12. Post-dialysis blood samples were collected at 3 and 6 months after entering this study. Results Serum folate and vitamin B12 remained unchanged during the study process. Serum folate was 5.16±0.87ng/ml, 4.94±0.85ng/ml and 4.75±0.77ng/ml at 0, 3 and 6 months, respectively, of high flux dialysis. However, the declining trend of serum folate after high flux dialysis was statistically insignificant (P＞0.05). Serum vitamin B12 level was 419.13±136.83pg/ml, 414.00±153.16pg/ml and 432.83±139.12pg/m, at 0, 3 and 6 months, respectively, of high flux dialysis (P＞0.05). Therefore, serum folate and vitamin B12 levels were unchanged before and after the dialysis. Conclusions Our results show that shifting from low flux to high flux hemodialysis does not result in the decrease of serum folate and vitamin B12.