The interaction between tetradecyl dimethyl benzyl ammonium chloride (TDBAC) and fatty alcohol polyoxyethylene ether (AEO-9) could significantly interfere with the ultraviolet absorption intensity of TDBAC in an aqueous solution. The results showed that AEO-9 can improve the ultraviolet absorption intensity of TDBAC and can greatly decrease the apparent critical micelle concentration (cmc) of TDBAC. When the concentration of AEO-9 increased from 0 to 0.150 and 0.300 mmol·L^-1, the apparent cmc of TDBAC decreased from 1.901 to 1.739 mmol·L^-1 and 1.584 mmol·L^-1, respectively. In TDBAC/AEO-9 aqueous solution, the ultraviolet absorption intensity of TDBAC was enhanced by adding 1∶1 β-cyclodextrin (β-CD) according to the molar amount of TDBAC, with the addition of 0.800 mmol·L^-1 β-CD, the ultraviolet absorption intensity of TDBAC increased from 0.259 to 0.270. When the concentrations of AEO-9 increased from 0 to 0.150 or 0.300 mmol·L^-1, TDBAC micelles could not be formed in the range of 0.600~2.800 mmol·L^-1 in aqueous solutionby adding 1∶1 β-CD. What is more, the interference of AEO-9 on UV Spectrum of TDBAC could be greatly reduced, and the recovery rate of TDBAC in TDBAC/AEO-9 aqueous solution changed from 86.3%~107.5% to 101.9%~103.9%, which showed that the detection accuracy of TDBAC was significantly improved. The results of Job’s experiments showed that TDBAC/AEO-9 inclusion should be formed with the molar ratio of 1∶1 in an aqueous solution. The results of FTIR andTG-DSC showed that TDBAC molecules were more likely to form inclusion complexes with β-CD rather than micelles in aqueous solution, which exhibited that the interference of AEO-9 on quantitative determination of TDBAC could be significantly reduced because of the formation of TDBAC/β-CD inclusion in TDBAC/AEO-9 aqueous solutions.