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Understanding the folding process of DNA origami is a critical stepping stone to the broader implementation of nucleic acid nanofabrication technology but is notably nontrivial. Origami are formed by several hundred cooperative hybridization events-folds-between spatially separate domains of a scaffold, derived from a viral genome, and oligomeric staples. Individual events are difficult to detect. Here, we present a real-time probe of the unit operation of origami assembly, a single fold, across the scaffold as a function of hybridization domain separation-fold distance-and staple/scaffold ratio. This approach to the folding problem elucidates a predicted but previously unobserved blocked state that acts as a limit on yield for single folds, which may manifest as a barrier in whole origami assembly.