Abstract Background It is becoming increasingly important to understand the mechanism of regulatory elements on target genes in long-range genomic distance. 3C (Chromosome Conformation Capture) and its derived methods are now widely applied to investigate genome organizations and gene regulation. Digestion-Ligation-Only Hi-C (DLO Hi-C) is a new technology with high efficiency and effective cost for whole-genome chromosome conformation capture. Results Here, we introduce DLO Hi-C Tool, a flexible and versatile pipeline for processing DLO Hi-C data from raw sequencing reads to normalized contact maps and providing quality controls for different steps. It includes more efficient iterative mapping and linker filtering. We applied DLO Hi-C Tool to different DLO Hi-C datasets, and demonstrated its ability of processing large data in multi-threading. Conclusions DLO Hi-C Tool is suitable for processing DLO Hi-C and in situ DLO Hi-C datasets. It is convenient and efficient for DLO Hi-C data processing.