The mRNA expression of protein Up-Frameshift Suppressor 3 Homolog B (UPF3B) differ in different tumors. However, the clinical relevance of UPF3B in cancer patients, such as with prognosis, tumor stage, and levels of tumor-infiltrating immune cells remain unclear.We performed bioinformatics analysis of UPF3B with The Cancer Genome Atlas (TCGA) database (https://xenabrowser.net) and TIMER2.0 (Tumor Immune Estimation Resource 2.0, http://timer.comp-genomics.org/). UPF3B expression in 33 cancers versus counterpart normal tissues was analyzed using TCGA pan-cancer data. The influence of UPF3B in long-term prognosis was evaluated using Kaplan-Meier method, and the associations between UPF3B transcription levels and immune-related gene expression, immune cell infiltration, tumor microenvironment (TME) score are analyzed by spearman correlation analysis. Enrichment analysis of UPF3B was conducted using the R package "clusterProfiler."The transcriptional level of UPF3B was dysregulated in the human pan-cancer dataset. A significant correlation was found between the expression of UPF3B and the pathological stage of Esophageal Carcinoma (ESCA), Kidney Chromophobe (KIHC), Liver Hepatocellular Carcinoma (LIHC), and Skin Cutaneous Melanoma (SKCM). Multiple cancer types with high transcriptional levels of UPF3B were associated with a significantly worse prognosis. The functions of expressed UPF3B gene are primarily related to ubiquitin mediated proteolysis, cell cycle, and mRNA surveillance pathway. Our results also show that immune cells infiltration and immunosuppressive markers such as CTLA-4, PD-1 and PD-L1 significantly correlate with UPF3B expression.In the present study, we synthetically explored the expression status and prognostic significance of UPF3B, and the relationship with clinic characters and immune microenvironment across cancers. Our results may provide novel insights for UPF3B as an immunotherapeutic target and valuable prognostic biomarker in various malignant tumor.