Recent advances in single-cell RNA sequencing (scRNA-seq) have improved our understanding of the association between tumor-infiltrating lymphocyte (TILs) heterogeneity and cancer initiation and progression. However, studies investigating alternative splicing (AS) as an important regulatory factor of heterogeneity remain limited. Here, we developed a new computational tool, DESJ-detection, which accurately detects differentially expressed splicing junctions (DESJs) between cell groups at the single-cell level. We analyzed 5063 T cells of hepatocellular carcinoma (HCC) and identified 1176 DESJs across 11 T cell subtypes. Interestingly, DESJs were enriched in UTRs, and have putative effects on heterogeneity. Cell subtypes with a similar function closely clustered together at the AS level. Meanwhile, we identified a novel cell state, pre-activation with the isoform markers ARHGAP15-205. In summary, we present a comprehensive investigation of alternative splicing differences, which provided novel insights into T cell heterogeneity and can be applied to other full-length scRNA-seq datasets.