In this study, four methods (phenol-chloroform protocol, sodium iodide kit, QIAamp DNA Blood Mini Kit, and TIANamp Micro DNA Kit) were used to extract cell-free DNA (cfDNA) from cattle blood, and the yield and purity of cfDNA varied in four different methods from 0.36 to 0.84 ng/mL for yield and 0.67 to 1.80 (A260/A280) for purity. Compared with other methods, the TIANamp Micro DNA kit performed better in both cfDNA amount and purity (p < 0.05); furthermore, blood cfDNA levels were significantly increased in Holstein dairy cows under the influence of heat stress (p < 0.01) and mastitis (p < 0.0001), which showed a potential power to discriminate mastitis (AUC = 0.99, 95% CI = 0.97 to 1.00) or heat stress (AUC = 0.86, 95% CI = 0.73 to 0.98) in cows. In brief, we established a complete experimental system for the extraction of cfDNA from cattle blood based on the high-yielding method of the TIANamp Micro DNA Kit and showed the effect of mastitis and heat stress on cfDNA levels in cattle blood for the first time. Our findings suggested that cfDNA in cattle blood may be a useful marker to measure mastitis and heat stress in dairy cattle.