Circulating tumor cells (CTCs) from liquid biopsy have shown a strong correlation to the clinical outcome of cancer patients. The enumeration and cytological analysis of CTCs have attracted increasing efforts for cancer disease management amid immunotherapy and personalized medicine. However, both enumeration and cytological analysis are challenging due to the rarity of CTCs and the lack of integrated solutions for the minimal risk of cell loss in the course of CTC procurement. We report a simple microfluidic chip permitting a one-stop solution for streamlining the on-chip cell separation, capture, immunofluorescence assay and/or in situ culture of isolated cells devoid of risky manual steps. Our results showed effective trapping of single cells, doublets and cell lumps isolated from blood in the same device. On-chip immunostaining revealed normal cell morphology and the characterization of cell expansion uncovered an altered cell growth curve with a reduced lag phase as compared to the conventional culture despite closely matching cell growth rates. The cells were viable and functional for as long as 11 days inside our chip and cell migration was also readily observed, with lumps showing greater aggressiveness than single cells. With these results, we expect promising applications of our one-stop solution for liquid biopsy via CTCs.