Studies that screen for metabolites produced in ruminants are actively underway. We aimed to evaluate the metabolic profiles of five biofluids (ruminal fluid, serum, milk, urine, and feces) in dairy cow by using proton nuclear magnetic resonance (1H-NMR) and provide a list of metabolites in each biofluid for the benefit of future research. We analyzed the metabolites in five biofluids from lactating cows using proton nuclear magnetic resonance imaging; 96, 73, 88, 118, and 128 metabolites were identified in the five biofluids, respectively. In addition, 8, 6, 9, and 17 metabolites were unique to ruminal fluid, serum, milk, and urine, respectively. The metabolites present at high concentrations were: acetate, propionate, and butyrate in ruminal fluid; lactate, glucose, and acetate in serum; and lactose, guanidoacetate, and glucitol in milk. In addition, the following metabolites were present at high concentrations: hippurate, urea, and trimethylamine N-oxide in urine and acetate, propionate, and butyrate in feces. The score plots of the principal component analysis did not show clear distinctions among the five biofluid samples. The purpose of this study was to verify the ability of our metabolomics approaches to identify metabolites in the biofluids of dairy cows.