The uses of single-stranded DNA probes tethered to solid supports have gained interest in recent years due to the increasing number of applications these systems present. However, the traditional methods used to fabricate these monolayers are flawed. The synthesis and purification of multithiol headgroups for improvement upon ssDNA surface attachment for the fabrication of self-assembled monolayers on gold surfaces is described. Generation 4 polyamidoamine dendrons were conjugated to ssDNA oligomers in a one-to-one ratio and functionalized to provide multiple thiol groups for possible surface attachment. Modification of the dendrimer’s amine terminated periphery groups to thiols was confirmed using 1H NMR spectroscopy. Successful conjugation of ssDNA to dendrons was confirmed using gel electrophoresis. DNA/dendron conjugate monolayers were compared with two other methods of DNA monolayer fabrication that are established in the literature: single-thiol DNA attachment and a technique that involves building a monolayer sequentially using surface chemistry, referred to as the DNA/dendrimer monolayer. These monolayers were compared using Grazing Angle Infrared spectroscopy, electrochemistry in combination with fluorescence microscopy, and Surface Plasmon Resonance. The results of this study show that the DNA/dendron conjugates had a better probe strand distribution than the DNA/dendrimer monolayer and a similar distribution as the single-thiol monolayer. In addition, the conjugates outperformed the other monolayers in terms of stability, reproducibility, binding of complementary DNA, and limit of quantitation. The results displayed in this study indicate that our novel DNA/dendron conjugates show promise as potential replacements in the making of DNA monolayers on gold substrates.