This study found distribution of polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans (PCDD/Fs) and dioxin like polychlorinated biphenyls (DL-PCBs) in bovine milk and ash samples collected from 9 different major districts namely Erode, Salem, Namakkal, Coimbatore, Trippur, Madurai, Chennai, Dindigul and Tiruchirappalli, which were highly populated and industrialized districts of South India (Tamil Nadu). The total toxic equivalency (TEQ) for PCDD/Fs and DL-PCBs in the bovine milk samples were ranged from 0.028 to 7.331-pg TEQ/g fat. Some of the districts showed higher PCDD/Fs and DL-PCBs concentration in milk and ash samples. Further, BM14 (7.331 pg-TEQ/g fat) and BM21 (6.406 pg-TEQ/g fat) sampling sites showed exceed level of PCDD/Fs and DL-PCBs than WHO regulation limits (6 pg-TEQ/g fat). Similarly, Total dioxins and DL-PCBs concentration in the ash sample were between 0.003-1. ng TEQ/g. Samples from AS3 (1.2 ng-TEQ/g) and AS11 (1.06 ng-TEQ/g) showed higher total dioxins and DL-PCBs level among other sampling sites. This study provides an overview of dioxins and dioxin-related compounds contamination in bovine milk and ash samples in south Tamil Nadu. Further, the CALUX assay method validation has simplified the monitoring of dioxin contamination in the environment.