Direct monitoring of single-molecule reactions has recently become a promising means of mechanistic investigation. However, the resolution of reaction pathways from single-molecule experiments remains elusive, primarily because of interference from extraneous processes such as bulk diffusion. Herein, we report a single-molecule kinetic investigation of DNA hybridization on a metal surface, as an example of a bimolecular association reaction. The tip of the scanning tunneling microscope (STM) was functionalized with single-stranded DNA (ssDNA), and hybridization with its complementary strand on an Au(111) surface was detected by the increase in the electrical conductance associated with the electron transport through the resulting DNA duplex. Kinetic analyses of the conductance changes successfully resolved the elementary processes, which involve not only the ssDNA strands and their duplex but also partially hybridized intermediate strands, and we found an increase in the hybridization efficiency with increasing the concentration of DNA in contrast to the knowledge obtained previously by conventional ensemble measurements. The rate constants derived from our single-molecule studies provide a rational explanation of these findings, such as the suppression of DNA melting on surfaces with higher DNA coverage. The present methodology, which relies on intermolecular conductance measurements, can be extended to a range of single-molecule reactions and to the exploration of novel chemical syntheses.