Heterogeneity in single cells has important implications for embryo development, neurogenesis and cancer progress. Single-cell transcriptome sequencing, decoding transcript sequences within cells based on high-throughput sequencing technology, can provide reliable and accurate research methods for cellular heterogeneity study to reveal the rule of biological development and disease progress. Different methods in single-cell transcriptome sequencing, single-cell multi-omics sequencing and spatial transcriptome analysis in recent years are summarized in this review. Challenges and perspectives of single-cell and spatial transcriptome analysis are discussed. Low-cost, all-round, high-throughput, high-resolution single-cell spatially multi-omics analysis will help to construct comprehensive and accurate single-cell atlas.