Abstract: A method for separation and determination of nonionic surfactant substances was established by liquid chromatography tandem mass spectrometry (LC-MS/MS). These nonionic surfactant substances contained dodecyl alcohol polyoxyethylene ethers, cetyl alcohol polyoxyethylene ethers and octadecyl alcohol polyoxyethylene ethers mixtures.The mass spectrometry of seven fatty alcohol polyoxyethylene ethers was analyzed, obtaining the mass spectrometry rule of such substances and deducing 41 fatty alcohol polyoxyethylene ethers components. This method was verified by neutral loss mass, parent scanning and daughter scanning. It carried out mass spectrum analysis and fragmentation regularity of such substances by positive ion electrospray ionization(ESI) scan mode. A simple and fast method was established to separate and identify different carbon chains and different addition of fatty alcohol polyoxyethylene ethers mixtures. This study can provide testing basis for this kind of materials determination in environmental water.