Single-cell sequencing has provided precision medicine with a new perspective for cancer detection because of its power to reveal cellular heterogeneity, especially in tumorigenesis. The application of next-generation sequencing (NGS) technique in circulating tumor cells (CTCs) further enhances the capability of cancer diagnosis and monitoring through liquid biopsy. Emerging technological developments in CTC enrichment and isolation, whole-genome amplification, single-cell sequencing analysis, and their applications on translational research and clinic practice are summarized in this chapter. The review also discusses the technical challenges in transforming CTC information and single CTC sequencing platform from academic research to practical use. Critical research questions are also highlighted in order to help provide clinical benefits for cancer patients using CTC-based single-cell sequencing techniques.