The study is aimed to detect and PCR characterize S. aureus enterotoxin gene from mastitic cow milk within Zaria metropolis. Four hundred and three quarter milk samples were collected from 104 lactating cows in 17 dairy hreds which included Palladan, Shika, Hanwa, Wusasa and Bomo. The physicochemical characteristics (Temperature, PH, and Specific gravity and Titratable acidity) and proximate composition (Moisture content, Protein content, Fat content and Ash content of the milk were determined. The prevalence of subclinical mastitis using California mastitis test (CMT), bacteriological analysis using standard methods, antibacterial susceptibility and identification of the gene encoding staphylococcal enterotoxin A and B using PCR technique were carried out. The Temperature, PH, Specific gravity and Titratable acidity of the milk ranged from 23-370C, 4.10 – 6.97, 1.023 – 1.032 and 0.14 – 0.23 respectively. The Moisture, Ash, Fat, and Protein content of the milk ranged from 86.99 – 87.67%, 0.66 – 0.69%, 3.22 – 3.81% and 3.13 – 3.45%. The prevalence of subclinical mastitis based on CMT reaction was (23.3%), 54 S.aureus were isolated from mastitic milk. The mean standard deviation ± of the total bacterial count from all the locations ranged from 6.22 ± 0.10 - 6.43 ± 0.12 and for the staphylococcal count from 2.98 ± 0.13 - 3.24 ± 0.11. The antibacterial susceptibility results shows that all the 54 S. aureus isolates were sensitive to vancomycin, norfloxacin and erythromycin (100%), 88.9% were sensitive to gentamicin and ciprofloxacin while 7.4% were sensitive to penicillin and methicillin. Out of the 15 isolates tested for enterotoxin A and B, 2 harboured gene for SEA and 5 isolates harboured gene for SEB but none of the isolates harboured both gene for SEA and SEB. The study concludes that further attention is needed to improve the hygienic and safety of milk and dairy farmers should be educated on the need to control mastitis, as it may decrease milk production and reduce the quality of the milk.