Menopause is an endocrinological transition that greatly affects health and disease susceptibility in middle-aged and elderly women. To gain new insights into the metabolic process of menopause, plasma metabolic profiles in 60 pre- and post-menopausal women were systematically analysed by biochemical methods in conjunction with univariate and multivariate statistical analysis. Biochemical markers signatures revealed considerable differences between pre- and post-menopausal women, and clear separations were observed. In total, five metabolites were identified as potential biochemical markers for menopause, including serum calcium, phosphorus, vitamin D, serum alkaline phosphatase and osteocalcin. These differences highlight those significant alterations occur in serum metabolism, biochemical reactions, hormone imbalance in post-menopausal women. In conclusion, our plasma biochemical study provides novel understanding of the metabolic profiles related to menopause, and will be useful for investigating menopause-related diseases and assessing metabolomic confounding factors.