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The modulation of steroid hormones on brain is attracting more and more attention. However, the detection of steroid hormones is faced with great challenge owning to the very low content in brain tissue. In this work, the preparation conditions of steroid hormones extracted from brain tissue were optimized. Ethanol was the best extraction solvent for brain homogenization as it readily penetrated cell membranes and was a good solvent for steroid hormones. After purified by Oasis HLB SPE, the matrix effect was reduced significantly. An on-line capillary solid phase extraction -nano liquid chromatographic system was built based on valve-switching 75 mu m i. d. trap column and analytical column. Tandem mass spectrometer with NanoESI source was used as the detector for the determination of ten brain steroid hormones. The method showed good precision (intraday RSD 4. 5% -12. 8% and inter day RSD 8. 3% -20. 3%) and linearity (r>0. 995). The limits of detection in solution were from 20 ng/L to 10 mu g/L for 10 steroid hormones. The sensitivity of the method was gained in two orders of magnitude compared with that of conventional HPLC/MS when the equal amounts of the steroid hormones were injected. The recoveries were above 60% at three concentrations except for androsterone while the matrix effect was inapparent except for progesterone. Brain tissue samples from mouse were analyzed using the new method. Four steroids were identified and quantified including testosterone 0. 47 ng/g, corticosterone 26. 2 ng/g, aldosterone 0. 49 ng/g and hydrocortisone 0. 06 ng/g. The results showed the method has many advantages such as high sensitivity, low sample amount and solvent consumption which is suitable for the determination of steroid hormones in biological tissue.