Organic thin film transistors (OTFTs) are excellent candidates for the application on disposable sensors due to their potentially low-cost fabrication process. A novel DNA sensor based on OTFTs with semiconducting polymer poly(3-hexylthiophene) has been fabricated by solution process. Both single- and double-strand DNA molecules are immobilized on the surface of the Au source/drain electrodes of different OTFT devices, producing a dramatic change in the performance of the devices, which is attributed to the increase of the contact resistances at the source/drain electrodes. Single-strand DNA and double-strand DNA are differentiated successfully in the experiments indicating that this is a promising technique for sensing DNA hybridization without labelling.