This study developed a single-molecule-based assay to track the looping (circularization) of individual double-stranded DNA (dsDNA) molecules in real time. DNA encodes our genetic information through a unique combination of four nucleotides: Adenine (A), Thymine (T), Cytosine (C), and Guanine (G). Base pairing among these nucleotides forms dsDNA molecules in a stable double-helical form. In addition to forming stable dsDNA, the genome must also achieve a precise three-dimensional architecture through twisting and bending of dsDNA; however, the mechanical properties (i.e., bending propensity) of dsDNA is sensitive to sequence, such that sequence defects can alter genome topology, resulting in genomic misfolding that has been linked to many disorders like cardiovascular diseases, cancers, schizophrenia, and limb development disorders. Looping is one of the fundamental structural transitions for organizing dsDNA. Using synthetic biology approaches, modified dsDNA molecules were designed and constructed that permit observation of dsDNA looping behavior by fluorescence microscopy. The assay shows that DNA looping is a highly dynamic process and that it is sensitive to ionic conditions and molecular crowding. Future work will probe how defects alter the looping behavior.