Objective  To explore the predictive value of serum levels of upstream stimulatory factor 2 (USF2), thioredoxin interacting protein (TXNIP), and Rho-associated coiled-coil containing protein kinase 2 (ROCK2) for the prognosis of patients with chronic renal failure (CRF) on hemodialysis.  Methods  A total of 85 CRF patients treated with hemodialysis for the first time in Cangzhou Hospital of Integrated TCM-WH Hebei from January 2021 to December 2022 were recruited as the research subjects. Enzyme linked immunosorbent assay (ELISA) was used to determine serum levels of USF2, TXNIP, and ROCK2. Logistic regression was used to explore the factors affecting prognosis of the CRF patients. Receiver operating characteristic (ROC) curve was used to assess the predictive value of serum levels of USF2, TXNIP, and ROCK2 for prognosis of the CRF patients. Moreover, Hosmer-Lemeshow (H-L) test was used for internal verification of the predictive models.  Results  Among the 85 CRF patient after hemodialysis treatment, 29 (34.12%) had a poorer prognosis, and 56 (65.88%) had a better prognosis. Compared with the better prognosis group, the poorer prognosis group had higher levels of serum USF2, TXNIP, and ROCK2 (t=6.603, 7.230 and 8.731 respectively; P＜0.001). Serum USF2 (HR=2.914, 95% CI: 1.365～6.222, P=0.006), TXNIP (HR=3.209, 95% CI: 1.717～5.997, P＜0.001), and ROCK2 (HR=2.135, 95% CI: 1.268～3.596, P=0.004) were the risk factors affecting prognosis of the CRF patients. The combination of serum USF2, TXNIP, and ROCK2 had higher area under the curve (AUC) than USF2, TXNIP or ROCK2 for predicting prognosis of the CRF patients (Zcombination-USF2=3.088, P=0.002; Zcombination-TXNIP=2.402, P=0.016; Zcombination-ROCK2=2.872, P=0.004).  Conclusion  Serum levels of USF2, TXNIP, and ROCK2 have a better predictive value for the prognosis of CRF patients on hemodialysis.