A real-time label-free DNA biosensor based on thin-core fiber (TCF) interferometer is demonstrated experimentally. The proposed biosensor is constructed by splicing a TCF between two segments of single mode fibers (SMFs) and integrated into a microfluidic channel. By modifying the TCF surface with monolayer poly-l-lysine (PLL) and single-stranded deoxyribonucleic acid (ssDNA) probes, the target DNA molecules can be captured in the microfluidic channel. The transmission spectra of the biosensor are measured and theoretically analyzed under different biosensing reaction processes. The results show that the wavelength has a blue-shift with the process of the DNA hybridization. Due to the advantages of low cost, simple operation as well as good detection effect on DNA molecules hybridization, the proposed biosensor has great application prospects in the fields of gene sequencing, medical diagnosis, cancer detection and environmental engineering.