Cerebrosides from sea cucumber Parastichopus californicus were identified by using liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-Q-TOF MS/ MS). The samples were extracted with chloroform-methanol (2: 1, v/v) solution and purified by a SPE cartridge. In positive ion mode electrospray ionization (ESI), the precursor ion scan mass spectra and product ion scan mass spectra were obtained through the automatic MS/MS mode. Cerebroside molecules were selected according to the neutral loss fragments of 180 Da, and then were identified according to long-chain base (LCB) fragments and fatty acid (FA) fragments. One hundred and twenty-three cerebroside molecular species were identified. There are 18 species of LCB, and the relative content ratio of phytosphingosines and sphingosines is 1: 2. The carbon numbers of fatty acids are mainly 18 - 25, of which 24 carbon fatty acids are predominant. The relative content ratio of saturated fatty acids and monounsaturated fatty acid is about 1: 3, and the presence of 2-hydroxy fatty acids is about 58.62%. LC-Q-TOF MS/MS method is sensitive, accurate and simple. At the same time, this study provided a theoretical basis for structure-activity relationship studies and functional food development of Parastichopus californicus as well.