Single-cell RNA sequencing (scRNA-seq) provides a comprehensive understanding of cellular complexity; however, its requirement for fresh or frozen samples limits its flexibility. To overcome this limitation to effectively leverage clinical samples, Chromium Fixed RNA Profiling on formalin-fixed paraffin-embedded (FFPE) tissue blocks (scFFPE-seq) was developed to perform single-nucleus RNA sequencing from nuclei isolated from FFPE. In this study, we utilized fresh tissue samples from colon, ileum, and skin to assess the viability of scFFPE-seq compared to these fresh samples. We were able to recover unique cell types from challenging FFPE tissues and validated scFFPE-seq findings through Hematoxylin and Eosin (H&E) images. The results demonstrated that scFFPE-seq effectively captured the single-cell transcriptome in FFPE tissues, obtaining comparable cell abundance, cell type annotation, and pathway characterization to those in fresh tissues. Overall, the study presents strong evidence of the potential of scFFPE-seq to enhance scientific knowledge by enabling the generation of high-quality, sensitive single-nucleus RNA-seq data from preserved tissue samples. This technique unlocks the vast archives of FFPE samples for extensive retrospective genomic studies.