The mechanical properties of a small fragment (30 bp) of an individual double-stranded deoxyribonucleic acid (dsDNA) in water have been investigated by atomic force microscopy (AFM). We have stretched three systems including ssDNA, double-fixed dsDNA (one strand of the dsDNA molecules was biotinylated at the 3'-end and thiolated at the 5'-end, this was reversed for the other complementary strand) and single-fixed dsDNA (one strand of the dsDNA molecules was biotinylated at the 3'-end and thiolated at the 5'-end, whereas the other complementary strand was biotinylated at only the 5'-end). The achieved thiolation and biotinylation were to bind ds- or ssDNA to the gold surface and streptavidin-coated AFM tip, respectively. Analysis of the force versus displacement (F-D) curves from tip-DNA-substrate systems shows that the pull-off length (L(o)) and stretch length (delta) from the double-fixed system were shorter than those observed in the ssDNA and the single-fixed system. The obtained stretch force (F(st)) from the single-fixed dsDNA was much greater than that from the ssDNA even though it was about 10 pN greater than the one obtained in the double-fixed system. As a result, the Young's modulus of the double-fixed dsDNA was greater than that of the single-fixed dsDNA and the ssDNA. A more reliable stiffness of the dsDNA was observed via the double-fixed system, since there is no effect of the unpaired molecules during stretching, which always occurred in the single-fixed system. The unpaired molecules were also observed by comparing the stiffness of ssDNA and single-fixed dsDNA in which the end of one strand was left free.