Bile acids (BAs) are a group of important physiological agents for cholesterol metabolism, intestinal nutrient absorption, and biliary secretion of lipids, toxic metabolites, and xenobiotics. Extensive research in the last two decades has unveiled new functions of BAs as signaling molecules and metabolic regulators that modulate hepatic lipid, glucose, and energy homeostasis through activation of nuclear receptors and G protein-coupled receptor signaling in gut-liver metabolic axis involving host-gut microbial co-metabolism. Therefore, investigation of serum BA profiles, in healthy human male and female subjects with a wide range of age and body mass index (BMI), will provide important baseline information on the BA physiology as well as metabolic homeostasis among human subjects that are regulated by two sets of genome, host genome and symbiotic microbiome. Previous reports on age- or gender- related changes on BA profiles in animals and human showed inconsistent results and the information acquired from various studies was highly fragmentary. Here, we profiled the serum BAs in a large population of healthy participants (n = 502) and examined the impact of age, gender and BMI on serum BA concentrations and compositions using a targeted metabonomics approach with ultra-performance liquid chromatography triple quadrupole mass spectrometry. We found that the BA profiles were dependent on gender, age, and BMI among study subjects. The total BAs were significantly higher in males than in females (p < 0.05) and higher in obese females than in lean females (p < 0.05). The difference in BA profiles between male and female subjects was decreased at age of 50-70 years, while the difference in BA profiles between lean and obese increased for subjects aged 50-70 years. The study provides a comprehensive understanding of the BA profiles in healthy subjects, and highlights the need to take into account age, gender and BMI differences when investigating pathophysiological changes of BAs resulting from gastrointestinal diseases.