Abstract: The BH4 and its precursors dihydroneopterin triphosphate and pyruvoyltetrahydropterin in the oleaginous fungus Mortierella alpina had been extracted and determined by high performance liquid chromatography (HPLC) with fluorescence detection.Mortierella alpina was triturated under liquid nitrogen and oxidized by I2KI under acidic condition for 1h.Then the sample was purified by ion exchange chromatography and analyzed by HPLC-fluorescence detection and electrospray ionization-mass spectrometry (ESI-MS) .HPLC was performed on an Inertsil ODS-3 C ₍18_ column (5μm, 150mm×4.6mm) .Aliquots of reaction mixtures were injected into the column and eluted by 10mmol/L Na₂HPO₄ (pH6.0) at a flow rate of 1.2mL/min.The excitation and emission wavelengths were set at 350nm and 450nm, respectively.The detection limit of this method for neopterin, pterin and biopterin were 0.003, 0.002 and 0.005μg/mL, respectively.The method was accurate and rapid, which could lay the foundation for the production of BH4 by Mortierella alpina.