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Background Primary liver cancer, predominantly hepatocellular carcinoma (HCC), has limited therapeutic options. While mutation-derived neoantigen vaccine holds promise, its success is hindered by low antigen availability. This study explores transcriptome-derived neoantigens (neoantigen-encoding tumor-specific transcripts, neoTSTs) in HCC, characterizing their features, generation mechanisms, and therapeutic potential.Methods We developed a computational pipeline integrating STAR/StringTie-based transcript assembly with multiexon/single-exon reference datasets (23,972 human control samples) for tumor-specific transcripts (TSTs) identification. A custom sliding-window algorithm compared TST-encoded peptides against UniProt, with neoTSTs predicted using netMHCPan. This framework was applied to 1,013 patients with liver cancer. NeoTSTs were validated through proteomics, immunopeptidomics, and HLA-transgenic models. Multiomics analyses characterized splicing patterns, transposable elements, and transcription factor regulation. Single-cell RNA-seq and Hep53.4 murine models assessed tumor coverage and immunotherapeutic efficacy.Results We analyzed RNA-seq data from 1,013 patients with liver cancer and constructed a multilayered reference dataset. Using a customized pipeline, we identified an average of 60 neoTSTs per patient, significantly surpassing mutation-derived neoantigens (neoMuts). NeoTSTs exhibited higher population frequencies, with 73.1% providing multiple epitopes, and were validated through mass spectrometry and HLA transgenic mouse models. Mechanistically, neoTSTs were generated via retained introns, transposable element activation, HNF4A-regulated alternative promoters, and de novo transmembrane domain generation. Single-cell analysis revealed neoTSTs cover >75% of tumor cells and identified antigen-presenting cancer-associated fibroblasts that enriched in immunotherapy responders and amplified CD4 + T-cell responses. In murine HCC models, neoTST vaccination outperformed neoMuts, inducing dual major histocompatibility complex-I/II activation and significant tumor growth inhibition.Conclusions NeoTSTs represent a superior neoantigen source in HCC, compensating for the limitations of mutation-derived targets. The remarkable abundance and patient-to-patient sharedness of neoTSTs underscore their dual potential: (1) as personalized immunotherapeutic targets, and (2) as broadly applicable antigens for low-TMB tumors. These findings provide a transformative framework for expanding treatment options in HCC immunotherapy.