To determine the expression of long non-coding RNA urothelial cancer-associated 1 (UCA1) by performing array-based quantitative polymerase chain reaction (PCR) and to identify the clinicopathological significance of UCA1 expression in prostate cancer using in situ hybridization (ISH) of surgically resected specimens. Array-based quantitative PCR was performed using 10 pairs of fresh malignant (prostate cancer) and normal tissue samples to determine UCA1 expression. Single-color RNA ISH of surgically resected prostate cancer specimens was performed using 70 formalin-fixed, paraffin-embedded tissue specimens to examine the clinicopathological significance of UCA1. Prostate cancer tissues exhibited higher levels of UCA1 expression than paired benign tissues. Furthermore, a correlation between high UCA1 expression and unfavourable clinicopathological characteristics, including advanced pathologic T stage, extraprostatic extension, presence of Gleason pattern 5, and involvement of the resection margins was observed. Notably, increased UCA1 expression significantly correlated with high- or very-high-risk patients, as defined by the 2023 National Comprehensive Cancer Network guidelines. UCA1 could be used as a novel diagnostic and prognostic biomarker for establishing an effective treatment protocol for prostate cancer.