Behavioral expressions and biochemical composition of body exudates are significantly altered in concert with the endocrine status, which are all clear indicators of physiological conditions of animals. In this study, we sought to infer about the reproductive physiological status of Kangayam cattle (Bos indicus) by analyzing behaviors, endocrine pattern, and body exudates and further to discover estrous biomarkers so as to facilitate timely artificial insemination/mating and to aid in aspects of conservation of the species. Therefore, in this study, we followed Kangayam cows through pre-estrous to post-estrous phases to correlate the endocrine dependence of biochemical constituents in urine and cervical mucus and sought to identify estrous biomarkers. Behavioral estrus was confirmed in 10 cows, from which urine samples were collected and subjected to determination of LH, FSH, estrogens, progesterone, proteins, and lipids. Furthermore, urinary fatty acids and proteins were profiled using gas chromatography and SDS-PAGE, respectively. The volatile compounds in the urine and cervical mucus were identified by gas chromatography-mass spectrometry analysis. The data revealed that LH, FSH, and estrogen levels increased significantly in estrous urine compared with nonestrous urine, whereas progesterone status was vice versa (P < 0.05). The lipid content was also significantly higher in estrous urine than in pre- and post-estrous urines (P < 0.05). There were also cyclical variations of volatiles and fatty acid profiles across phases of the estrous cycle. More acidic compounds were present in estrous urine, rendering it more acidic, than in pre- and post-estrous urines. Interestingly, oleic acid, which was present as a fatty acid in estrous and post-estrous urines, appeared to be a volatile in post-estrous urine and estrous cervical mucus. In addition, octanoic and butanoic acids were specific to both estrous urine and cervical mucus, indicating their possible candidature as estrous biomarkers. SDS-PAGE analysis showed pronounced expression of a 98 kDa protein in post-estrous urine, which in matrix-assisted laser desorption ionization-time of flight mass spectrometry was identified as albumin. Our results demonstrate multiple biomarkers in estrous urine and specific volatiles in cervical mucus that offer scope to develop viable estrus detection kits for Kangayam cows.