In recent years, the development of spatial transcriptomic technologies has enabled us to gain an in-depth understanding of the spatial heterogeneity of gene expression in biological tissues. However, a simple and efficient tool is required to analyze multiple spatial targets, such as mRNAs, miRNAs, or genetic mutations, at high resolution in formalin-fixed paraffin-embedded (FFPE) tissue sections. In this study, we developed hydrogel pathological sectioning coupled with the previously reported Sampling Junior instrument (HPSJ) to assess the spatial heterogeneity of multiple targets in FFPE sections at a scale of 180 μm. The HPSJ platform was used to demonstrate the spatial heterogeneity of 9 ferroptosis-related genes (TFRC, NCOA4, FTH1, ACSL4, LPCAT3, ALOX12, SLC7A11, GLS2, and GPX4) and 2 miRNAs (miR-185–5p and miR522) in FFPE tissue samples from patients with triple-negative breast cancer (TNBC). The results validated the significant heterogeneity of ferroptosis-related mRNAs and miRNAs. In addition, HPSJ confirmed the spatial heterogeneity of the L858R mutation in 7 operation-sourced and 4 needle-biopsy-sourced FFPE samples from patients with lung adenocarcinoma (LUAD). The successful detection of clinical FFPE samples indicates that HPSJ is a precise, high-throughput, cost-effective, and universal platform for analyzing spatial heterogeneity, which is beneficial for elucidating the mechanisms underlying drug resistance and guiding the prescription of mutant-targeted drugs in patients with tumors.