Molecularly directed self-assembly has the potential to become a nanomanufacturing technology if the critical factors governing the kinetics and yield of defect-free self-assembled structures can be understood and controlled. The kinetics of streptavidin-functionalized quantum dots binding to biontinylated DNA origami are quantitatively evaluated and to what extent the reaction rate and binding efficiency are controlled by the valency of the binding location, the biotin linker length, and the organization, and spacing of the binding locations on the DNA is shown. Yield improvement is systematically determined as a function of the valency of the binding locations and as a function of the quantum dot spacing. In addition, the kinetic studies show that the binding rate increases with increasing linker length, but that the yield saturates at the same level for long incubation times. The forward and backward reaction rate coefficients are determined using a nonlinear least squares fit to the measured binding kinetics, providing considerable physical insight into the factors governing this type of self-assembly process. It is found that the value of the dissociation constant, Kd, for the DNA–nanoparticle complex considered here is up to seven orders of magnitude larger than that of the native biotin–streptavidin complex. This difference is attributed to the combined effect that the much larger size of the DNA origami and the quantum dot have on the translational and rotational diffusion constants.