Hepatocellular carcinoma (HCC) represents a significant health concern. Identifying novel molecular targets is crucial for clinical diagnosis and targeted treatment of HCC. Aptamers are capable of binding specifically to cancer cells via target protein molecules. Consequently, aptamers are frequently employed to identify novel cancer biomarkers. The invasiveness of tumor cells is closely associated with the recurrence and metastasis of tumors. In this study, the highly invasive Huh7-P3 cells were initially constructed, and subsequently, several aptamers that could specifically recognize Huh7-P3 were developed using cell-based Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The selected aptamer, designated S2-2, demonstrated the capacity to bind to multiple cancer cells. Furthermore, tissue imaging demonstrated that S2-2 exhibited a specific recognition of HCC tissue, while demonstrating no binding to normal tissue. Subsequently, voltage-dependent anion channel 1 (VDAC1) was identified as a potential target for S2-2. Furthermore, Doxorubicin (Dox)-loaded S2-2 was shown to specifically kill target Huh7-P3 cells. In vivo fluorescence imaging revealed that S2-2 was capable of specifically targeting tumors. Importantly, S2-2-Dox enhanced the anti-tumor efficacy of Dox in CDX model. This study may provide a promising biomarker and molecular target for the clinical diagnosis and targeted therapy of cancers with high VDAC1 expression.