We proposed and experimentally demonstrated an optofluidic biosensor using a twin-core hollow optical fiber (TC-HOF) interferometer for label-free sensing of the streptavidin-biotin binding. The TC-HOF had a central hole and two cores were imbedded near its circumference. The central hole functioned as a microfluidic channel where the liquid was flown. One of the cores was suspended at the inner surface of the hole and served as sensing arm while the other core embedded in the cladding as the reference arm, which formed an integrated all-fiber Mach-Zehnder interferometer (MZI). Both ends of TC-HOF were serially spliced to multimode fiber-single mode fiber assembly, whose lateral positions were adjusted to maximize the interference visibility. Before the surface functionalization, the pristine TC-HOF MZI showed the RI sensitivity of 2577 nm/RIU. The suspended core's surface was chemically modified with streptavidin and biotin solutions were injected through micro-HOles in TC-HOF. Through the specific binding reaction between biotin and streptavidin, the effective refractive index (RI) around the sensing arm was modulated, which was detected by the spectral shifts in the interference patterns. The proposed sensor showed an excellent linear response to the biotin concentration in the range of 0.01–0.1 mg/mL and the net detection sensitivity of biotin was 16.9 nm/(mg/mL), which was achieved in a label-free manner. This device can be further applied to sensing and monitoring of the biochemical binding of biotin with various biomolecules such as cells, antigens, antibodies, and DNA in real-time and in situ. © 2018