A fundamental understanding of molecular self-assembly processes is important for improving the design and construction of higher-order supramolecular structures. DNA tile based self-assembly has recently been used to generate periodic and aperiodic nanostructures of different geometries, but there have been very few studies that focus on the thermodynamic properties of the inter-tile interactions. Here we demonstrate that fluorescently-labeled multihelical DNA tiles can be used as a model platform to systematically investigate multivalent DNA hybridization. Real-time monitoring of DNA tile assembly using fluorescence resonance energy transfer revealed that both the number and the relative position of DNA sticky-ends play a significant role in the stability of the final assembly. As multivalent interactions are important factors in nature's delicate macromolecular systems, our quantitative analysis of the stability and cooperativity of a network of DNA sticky-end associations could lead to greater control over hierarchical nanostructure formation and algorithmic self-assembly.