To date, most studies have focused on the improvement of dairy nutritional management strategies to enhance milk production and quality, whereas the physiological and metabolic mechanisms involved have not been well examined. This study was conducted to investigate the relationships of 4 biofluids from lactating dairy cow to identify mechanisms and potential biomarkers as well as key pathways involved in potential regulation of lactation. Eight multiparous Holstein dairy cows with similar lactation stage (164 ± 19.6 d, mean ± SD) and milk yield (30.4 ± 2.29 kg, mean ± SD) were offered a diet with 16.7% (DM basis) of crude protein and 1.57 Mcal/kg of net energy for lactation. Rumen fluid, serum, milk, and urine were collected after 65-d feeding to identify the metabolome using gas chromatography–time of flight/mass spectrometry. A total of 165, 195, 218 and 156 metabolites were identified from rumen fluid, milk, serum, and urine, respectively, with 29 metabolites detected in all 4 biofluids. The TIC chromatograms showed a clear discrimination among 4 biofluids; and principal component analysis of the relative concentration of mutual metabolites revealed 4 separated metabolite profile clusters of 4 biofluids. The clusters derived from the rumen fluid, milk, and serum partly overlapped with each other, whereas the cluster from the urine was separated from the other 3 biofluids. The dendrogram of hierarchical clustering analysis revealed different subclusters containing varying numbers of metabolites within each biofluid; and the subclusters from rumen fluid and serum were grouped together and highly correlated with each other, but were separated from milk. Based on metabolomic profiles, urine is the most different biofluid, compared with other 3 biofluids. When the mutual metabolites were used for pathway analysis, the impact values of the pathway were 0.29, 0.28, and 0.11 for glycine, serine and threonine metabolism, glycerolipid metabolism, and tyrosine metabolism, respectively. These 3 pathways may play important roles in improvement of lactation performance. Our results suggest that all the 4 biofluids represent the comprehensive metabolism of dairy cow that can be further used for metabolic pathway analysis.