Pancreatic cancer (PC) has the poorest prognosis compared to other common cancers because of its aggressive nature, late detection, and resistance to systemic treatment. In this study, we aimed to identify novel biomarkers for PC patients and further explored their function in PC progression. We analyzed GSE62452 and GSE28735 datasets, identifying 35 differentially expressed genes (DEGs) between PC specimens and non-tumors. Based on 35 DEGs, we performed machine learning and identified eight diagnostic genes involved in PC progression. Then, we further screened three critical genes (CTSE, LAMC2 and SLC6A14) using three GEO datasets. A new diagnostic model was developed based on them and showed a strong predictive ability in screen PC specimens from non-tumor specimens in GEO, TCGA datasets and our cohorts. Then, clinical assays based on TCGA datasets indicated that the expression of LAMC2 and SLC6A14 was associated with advanced clinical stage and poor prognosis. The expressions of LAMC2 and SLC6A14, as well as the abundances of a variety of immune cells, exhibited a significant positive association with one another. Functionally, we confirmed that SLC6A14 was highly expressed in PC and its knockdown suppressed the proliferation, migration, invasion and EMT signal via regulating Wnt/β-catenin signaling pathway. Overall, our findings developed a novel diagnostic model for PC patients. SLC6A14 may promote PC progression via modulating Wnt/β-catenin signaling. This work offered a novel and encouraging new perspective that holds potential for further illuminating the clinicopathological relevance of PC as well as its molecular etiology.