The interaction between nanoparticles (NPs) and DNA plays an important role in the genotoxicity of NPs, and it is imperative to characterize the nano/DNA interactions and explore the underlying chemical mechanisms. In this chapter, we demonstrated systematic experimental approaches based on atomic force microscope (AFM), coupled with modeling computation to probe the binding activity of NPs with DNA and the putative genotoxicity. Using quantum dots (QDs) as a model NP, we examined the binding kinetics, binding isotherm, binding specificity, and binding mechanisms of NPs to DNA with the application of AFM. We further assessed the binding affinity between NPs and DNA by calculating their interaction energy on the basis of Derjaguin-Landau-Verwey-Overbeek (DLVO) models. The modeling results of binding affinity were validated by the NPs/DNA binding images experimentally derived by AFM. The investigation of the relationship between the binding affinity of five NPs ((QDs (+), QDs (-), silver NPs, hematite NPs, and gold NPs) for DNA with their inhibition effects on DNA replication indicated that NPs with a high binding affinity for DNA molecules exhibited higher inhibition on DNA replication. The methodology employed in this study can be extended to study the interaction of other NPs with DNA, which is anticipated to benefit the future design of safe NPs, as well as the toxicological investigations of NPs.