As a new, water-soluble material, graphene oxide (GO) has gained growing interest for sensing applications. Particularly interesting is the interaction of nucleic acids with GO. Recently, it was found that short single-stranded DNA (ssDNA) had weaker affinity to GO than long ssDNA. This property makes it possible to prepare a novel bioassay platform for metal ions, antibiotics, and nuclease detection via the DNA(RNA) cleavage reaction. While practical analytical applications have been successfully demonstrated, few studies are focused on the mechanism of this phenomenon. In this work, we use fluorescence spectroscopy to deeply investigate the binding mechanism of ssDNA with GO to reveal the reason for this affinity difference caused by DNA length. Through computing with literature models, the main binding force, the binding constant, and number of binding sites between ssDNA and GO are obtained. Besides, our results show that the binding constant of short ssDNA with GO is much lower than that of long ssDNA with GO, which is the strongest evidence to prove the affinity difference between short ssDNA and long ssDNA with GO. Finally, based on these basic understandings of the interaction between ssDNA and GO, we develop a GO based biosensor for S1 nuclease and an inhibitor of S1 nuclease with satisfying results.