We describe LnCeCell 2.0 (http://bio-bigdata.hrbmu.edu.cn/LnCeCell), an updated resource for lncRNA-associated competing endogenous RNA (ceRNA) networks and web tools based on single-cell and spatial transcriptomics sequencing (stRNA-seq) data. We have updated the LnCeCell 2.0 database with significantly expanded data and improved features, including (i) 257 single-cell RNA sequencing and stRNA-seq datasets across 86 diseases/phenotypes and 80 human normal tissues, (ii) 836 581 cell-specific and spatial spot-specific ceRNA interactions and functional networks for 1 002 988 cells and 367 971 spatial spots, (iii) 15 489 experimentally supported lncRNA biomarkers related to disease pathology, diagnosis and treatment, (iv) detailed annotation of cell type, cell state, subcellular and extracellular locations of ceRNAs through manual curation and (v) ceRNA expression profiles and follow-up clinical information of 20 326 cancer patients. Further, a panel of 24 flexible tools (including 8 comprehensive and 16 mini-analysis tools) was developed to investigate ceRNA-regulated mechanisms at single-cell/spot resolution. The CeCellTraject tool, for example, illustrates the detailed ceRNA distribution of different cell populations and explores the dynamic change of the ceRNA network along the developmental trajectory. LnCeCell 2.0 will facilitate the study of fine-tuned lncRNA-ceRNA networks with single-cell and spatial spot resolution, helping us to understand the regulatory mechanisms behind complex microbial ecosystems.