Circulating tumor cells (CTCs) are an important biomarker for various types of cancers. As CTCs are extremely rare in the bloodstream, the accurate analysis and efficient separation of CTCs is a challenging task. In order to develop new diagnostic approaches for cancer research, understanding the functional characteristics of biomolecules at the single-molecule level in individual cancer cells is required. Meanwhile, microfluidic platforms have emerged as a promising technology for analyzing single molecules and single cells. In this review, we discuss some label-free techniques for biomolecule detection and highlight some reports that have employed combined label-free techniques with microfluidics for individual molecule or cancer cell analysis. Finally, we offer our perspective of the integration of label-free techniques with microfluidic technology on future applications.