Ubiquitination, a post-translational modification, refers to the covalent attachment of ubiquitin molecules to substrates. This modification plays a critical role in diverse cellular processes such as protein degradation. The specificity of ubiquitination for substrates is regulated by E3 ubiquitin ligases. Dysregulation of ubiquitination has been associated with numerous diseases, including cancers. In our study, we first investigated the protein expression patterns of E3 ligases across 12 cancer types. Our findings indicated that E3 ligases tend to be up-regulated and exhibit reduced tissue specificity in tumors. Moreover, the correlation of protein expression between E3 ligases and substrates demonstrated significant changes in cancers, suggesting that E3-substrate specificity alters in tumors compared to normal tissues. By integrating transcriptome, proteome, and ubiquitylome data, we further characterized the E3-substrate regulatory patterns in lung squamous cell carcinoma. Our analysis revealed that the upregulation of the SKP2 E3 ligase leads to excessive degradation of BRCA2, potentially promoting tumor cell proliferation and metastasis. Furthermore, the upregulation of E3 ubiquitin–protein ligase TRIM33 was identified as a biomarker associated with a favorable prognosis by inhibiting the cell cycle. This work exemplifies how leveraging multi-omics data to analyze E3 ligases across various cancers can unveil prognosis biomarkers and facilitate the identification of potential drug targets for cancer therapy.