Lactotransferrin (LTF), a critical multifunctional glycoprotein, plays an essential role in the immune defence, growth and development, and milk quality of dairy cows. The regulatory mechanisms governing gene expression are intricate, with sequence variations in the promoter region potentially exerting a substantial impact on gene expression. In this study, sequencing analysis of the bovine lactotransferrin promoter region was conducted, leading to the identification of two linked single nucleotide polymorphism (SNP) sites. A significant association between these SNPs and lactotransferrin content was observed in a cohort of 301 Holstein cows. Subsequently, further investigation into the transcriptional activity of various lactotransferrin genotypes was performed by constructing promoter fragments encompassing different lactotransferrin genotypes. The findings reveal that the two SNPs significantly influence the activity of the lactotransferrin promoter, thereby affecting lactotransferrin expression. These results hold substantial implications for advancing our understanding of the regulatory mechanisms underlying lactotransferrin expression and for the genetic enhancement of dairy cows.