DNA double-strand break (DSB) is the most dangerous type of DNA damage. In addition, DSBs are also common consequences of various therapeutic and genetic modifications. Therefore, quantification of DSB is of great importance in many fields including DNA damage repair, cancer therapy, gene editing, and radiation biology. Current methods are either low-throughput, laborious, or high cost. Here, we developed dc-BLIS (digital counting of breaks labeling in situ), a new method that can rapidly and precisely quantify the number of intracellular DSBs at a low cost by digital polymerase chain reaction. Using dc-BLIS, we quantified and compared the amount of DSBs induced by anti-cancer drugs, Cas9 variants, and different radiation doses, proving the capacity of dc-BLIS to quantify DSBs. We propose that dc-BLIS is suitable for various application scenes that require rapid and precise quantification of DSBs, including drug screening, gene-editing tool modification, and radiation effect assessment.