The fertility of dairy cows has been declining worldwide. The number of services per conception has increased, and repeat breeder (RB) cows are considered important in the dairy industry. However, there has been little research on RB cows in Korea. The objective of this study was to investigate the relationship between serum biochemical profiles and RB Holstein cattle in Korea and compare the results with those of studies conducted abroad. In addition, we investigated hidden factors that are needed for RB cattle to become pregnant. Overall, 34 Korean Holstein Friesian cows were divided into three groups: pregnant with normal cycling (PNC), pregnant with repeat breeder (PRB), and non-pregnant with repeat breeder (NRB). Blood samples were collected from the jugular vein, and serum parameters (ALT, AST, ALP, GGT, LD, CK, TP, ALB, GLOB, TBIL, BUN, CRE, GLU, TC, TG, NEFA, CA, MG, and IP) were analyzed. GGT levels were significantly different among groups (P=0.01). The concentration of GGT was the highest in the NRB and the lowest in the PNC. In contrast to the findings of other studies, there were no differences in GLU, TP, TC, and BUN levels. This study is the first to investigate serum biochemistry in RB cattle and provides results that differ from those of previous studies; these findings would help establish a novel approach to improve fertility of RB cattle.