Animal urine plays a very important role in animal life. The detection of chemical compounds in Tupaia belangeri urine is the key to identify its composition and assay the function of each chemical compound. In the present study, headspace-solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) were used to analyze the urine compositions in Tupaia belangeri. Through searching the mass spectrometry library of the National Institute of Standards and Technology (NIST), we found that the chemical compounds belong mainly to aldehydes, ketones, acids, esters, aromatic hydrocarbons, hydrocarbons, and phenols, etc. The results showed that HS-SPME-GC/MS is an effective method to detect volatile and semi-volatile compounds in tree shrew urine. In addition, wild individuals with relatively abundant food sources have more components than do those fed on an artificial diet., There are sex differences in the chemical compositions of urine. The results provide a foundation for studying the mechanism of chemical communication in tree shrews.