Tumor heterogeneity is a major hindrance in cancer classification, diagnosis and treatment. Recent technological advances have begun to reveal the true extent of its heterogeneity. Single‐cell analysis (SCA) is emerging as an important approach to detect variations in morphology, genetic or proteomic expression. In this review, we revisit the issue of inter‐ and intra‐tumor heterogeneity, and list various modes of SCA techniques (cell‐based, nucleic acid‐based, protein‐based, metabolite‐based and lipid‐based) presently used for cancer characterization. We further discuss the advantages of SCA over pooled cell analysis, as well as the limitations of conventional techniques. Emerging trends, such as high‐throughput sequencing, are also mentioned as improved means for cancer profiling. Collectively, these applications have the potential for breakthroughs in cancer treatment.