Hepatocellular carcinoma (HCC) is a common and high malignant tumor. It is a main subtype of primary liver cancer with a poor prognosis. Pseudouridine (Ψ) modification participates in the progression of various tumors, but its role in the malignant progression of HCC has not been explored. The prognostic value of pseudouridine synthases (PUSs) was investigated in 374 hepatocellular carcinoma (HCC) samples from The Cancer Genome Atlas (TCGA). We systematically assessed 13 pseudouridine modifications and comprehensively analyzed the differential expression in HCC. Cox regression analysis and a LASSO regression model were combined to build a prediction model composed of five genes (RPUSD4, PUS1, PUS7, RPUSD3, DKC1). We used the prediction model to divide the TCGA database into high-risk and low-risk subgroups in HCC. Then we identified the molecular characteristics, prognostic significance, infiltrating immune cell intensities of the identifying two high and low risk groups, predicted the clinical outcome of HCC patients and immunotherapeutic effect. At the same time, in the IMvigor 210 databas, patients with high-risk score showed better curative effect, which is consistent with the results of TIDE. Finally, we verified the expression of PUS1 and PUS7 in HCC tumors and nontumors by immunohistochemistry. In conclusion, our findings showed that a novel five-gene signature related to epigenetics can accurately predict the occurrence and prognosis of HCC. The protein expression of PUS1 and PUS7 is significantly different in HCC and normal tissues, which can be used as a key research object.