This paper deals with the separation and characterization of alkyl phenol formaldehyde resins demulsifier by infrared spectrometry and nuclear magnetic resonance spectroscopy after separation of the different surfactants and lowmolecular additives by adsorption chromatography. Firstly, the types of surfactants are identified by methylene blue chloride–chloroform test method and the elemental analysis such as Ca, K, Mg, Na, P, S and N. Then, the different surfactants and low molecular components are separated by adsorption chromatography after parts of low molecular components are dried in an oven, and the molecular weight distribution is measured by gel permeation chromatography also. Finally, the separated surfactants are determined by Fourier transform infrared (FTIR) spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The distortion less enhancement by polarization transfer (DEPT), H, C correlated spectroscopy (H, C–COSY), H, H correlated spectroscopy (H, H–COSY) and heteronuclear multiple-bond correlation (HMBC) spectroscopy are applied to determine the molecular structures.