It is hard to determine the concentration of sodium lauryl benzenesulfate (SDBS) or betaine in their complex system due to the strong interference between SDBS and betaine. A new method is developed for reducing the interactions between SDBS and betaine by adding β-cyclodextrin (β-CD) in their aqueous complex solution. Several factors such as the effect of β-CD on quantitative analysis of SDBS, the anti-interference ability of β-CD as well as the effect of interactions between SDBS and betaine are investigated. The results indicate that the effect of the interactions between SDBS and betaine on determination of SDBS/betaine complex system can be greatly reduced by adding β-CD, thus, the determination precision of SDBS and betaine can be greatly improved. The recovery rates of SDBS and betaine are respectively reduced from 103.9%~105.9% and 103.4%~174.9% to 101.8%~ 102.3% and 102.3%~102.8% in their aqueous complex solution. Besides, the experimental results further show that the SDBS and betaine complex in environmental water can also be analyzed by the method. The results of nuclear magnetic resonance hydrogen spectrum (1H-NMR) analysis and Fourier transform infrared spectroscopy (FT-IR) analysis show that the stale β-CD—SDBS inclusion can be formed by entering of SDBS molecules from the narrow or broad mouth of β-CD. The interference between SDBS and betaine can be reduced by β-CD because of the formation of β-CD and SDBS inclusion complex.