Human topoisomerase II alpha (TOP2α) resolves DNA intertwines between sister chromatids during mitosis. How cohesin, an SMC complex that holds sister chromatids, affects TOP2α decatenation is unclear. To addres this, we developed a quadruple-trap optical tweezers assay to create DNA braids and study TOP2α decatenation at the single-molecule level in real-time. We show that TOP2α resolves both single and multiple braids but becomes inefficient at forces exceeding 28 pN. TOP2α is sensitive to DNA geometry, exhibiting a chiral preference for right-handed braid crossings, and requires loading directly at DNA crossovers to act. Pre-loading TOP2α onto individual DNA strands before braid formation, in the presence of ATP, prevents decatenation. Finally, we show that human cohesin, but not condensin I, binds stably to DNA braids and blocks TOP2α activity. Our study provides novel insights into the role of substrate accessibility in regulating TOP2α‘s activity and highlights cohesin as a barrier to decatenation.