Abstract Objective This study aimed to investigate the diagnostic value of stress-induced phosphoprotein 1 (STIP1) in serum for hepatocellular carcinoma (HCC) and alpha-fetoprotein (AFP)–negative HCC (ANHC). Methods In this study, serum samples were collected from 158 HCC patients and 63 non-HCC patients. Logistic regression analysis was performed to identify independent risk factors associated with HCC and ANHC. The diagnostic values of each index for HCC and ANHC were analyzed using receiver operating characteristic (ROC) curve analysis. Results The STIP1, des-γ-carboxy prothrombin (DCP), and AFP levels were higher in the HCC groups than in the non-HCC groups (P < .05). Age, DCP, STIP1, and hepatitis B virus infection were independent predictors of HCC (P < .05). The diagnostic value of STIP1 for HCC was higher than that of DCP. Additionally, age, STIP1, and hepatitis B virus infection were independent predictors for ANHC patients. The ROC curve exhibited an area under the curve value of 0.919 for STIP1, with a diagnostic cutoff value of 68.5 U/mL. Moreover, 36 ANHC patients and 19 AFP-negative non-HCC patients were included to validate the diagnostic model. A total of 20 patients had STIP1 levels greater than 68.5 U/mL, resulting in diagnostic accuracy of 67.3%, sensitivity of 55.6%, and specificity of 89.5%. Conclusion STIP1 demonstrates excellent diagnostic value for HCC and ANHC.