DNA and carbon dots were utilized to co-mediate the growth of gold nanoparticles (AuNPs). The properties of grown AuNPs were discussed and applied to colorimetric biomolecule determination. When target biomolecules were absent, single-stranded oligonucleotides (ssDNA) were adsorbed on the surface of AuNP seeds. Branched AuNP solution of blue color was obtained after carbon dots reduction. In the presence of target DNA, it hybridized with the ssDNA and double-stranded oligonucleotides (dsDNA) were formed. As a result, a spherical AuNP solution of red color was achieved after growth as dsDNA would not be adsorbed by AuNP seeds. A similar situation was observed with thrombin. The complex of thrombin and its aptamer was formed, giving rise to spherical AuNPs after growth. The target biomolecule determination was realized by virtue of color change, consistent with the peak shift in UV–vis absorption spectrum. The detection limits were determined to be 5nM for target DNA and 1.8nM for thrombin, respectively. The biomolecule sensors showed the requisite selectivity against possible interferents, and the assay for target DNA exhibited the potential for distinguishing single-base mismatch. These results served as the basis for constructing future biomolecule sensors using DNA and carbon dots co-mediated growth of AuNPs.