A novel and highly sensitive fiber-optic DNA sensor based on a thin-core fiber modal interferometer (TCFMI) is demonstrated by using a layer-by-layer (LbL) self-assembly technology. Poly(ethylenimine) (PEI), poly(acrylic acid) (PAA) and single-stranded DNA (ssDNA) were used for the preparation of a polyelectrolyte multilayer film for DNA detection. The film thickness was measured through a surface profilometer. The surface morphologies of (PEI/PAA)4, (PEI/PAA)4.5 and (PEI/PAA)4(PEI/DNA)1 multilayer films were characterized by atomic force microscopy. The fabricated DNA sensors were tested with different types of target ssDNA solutions with a concentration of 1 μM. The results show that the sensitivity of the TCFMI-based ssDNA sensor is 0.27 nm/matched-base at the concentration of 1 μM and can even distinguish the number of matched bases of ssDNA chains.