Abstract Purpose: Positron emission tomography (PET) imaging was not efficiently used in early diagnosis of hepatocellular carcinoma (HCC) due to lack of appropriate tracers. Sodium pump Na+/K+ ATPase subunit α1 (NKAα1) emerges to be a prognostic and diagnostic biomarker of HCC. Here we investigated the feasibility of 18F-ALF-NOTA-S3, a PET tracer based on an NKAα1 peptide, to detect small HCC. Methods: GEPIA database was searched to obtain the expression characteristics of NKAα1 in HCC and its relationship with the prognosis. PET/CT was performed in orthotopic, diethylnitrosamine (DEN)-induced and genetically engineered HCC mouse models to evaluate the use of 18F-ALF-NOTA-S3 to detect HCC lesions. Results: NKAα1 is overexpressed in early HCC with a high positive rate and correlates with poor survival. In orthotopic, DEN-induced and genetically engineered HCC mouse models, PET/CT imaging showed high accumulation of 18F-ALF-NOTA-S3 in the tumor. The tumor-to-liver ratios are 2.56 ± 1.02, 4.41 ± 1.09 and 4.59 ± 0.65 respectively. Upregulated NKAα1 expression in tumors were verified by immunohistochemistry. Furthermore, 18F-ALF-NOTA-S3 has the ability to detect small HCC lesions with diameters of 2-5mm. Conclusion: NKAα1 may serve as a suitable diagnostic and prognostic biomarker for HCC. 18F-ALF-NOTA-S3 shows great potential for PET imaging of HCC.