In the binary mixtures of sodium dodecyl sulfate (SDS) and alkylphenol ethoxylates (NP-40), SDS can significantly affect the UV spectrum and critical micelle concentration (cmc) of NP-40. Although SDS has no obvious absorption peak in the range of 240 ~ 320 nm, SDS can enhance the absorbance of NP-40 at 275 nm. After adding 0.50 mmol×L^-1 SDS, the absorbance of 0.25 mmol×L^-1 NP-40 increased from 0.345 to 0.378, an increase of 9.6%. At the same time, SDS could also significantly reduce the apparent cmc of NP-40, when the concentration of SDS in aqueous solution was 0.20 mmol.L^-1 and 0.50 mmol.L^-1, the apparent cmc of NP-40 decreased from 0.237 mmol×L^-1 to 0.227 mmol.L^-1 and 0.224 mmol.L^-1, respectively. The results showed that hydroxypropyl-β-cyclodextrin (HP-β-CD) could effectively reduce the interference of SDS on the UV spectrum of NP-40. After HP-β-CD was added, the absorbances of 0.25 mmol.L^-1 NP-40 and its absorbance in 0.50 mmol.L^-1 SDS solution were 0.379 and 0.381, respectively. Compared with that without HP-β-CD, the absorbance change was reduced from 9.6% to 0.8%. The results also showed that HP-β-CD could hinder the formation of NP-40 micelles, and the Δ_rG_m^θ of NP-40 micelles increased with the increase of HP-β-CD concentration. After HP-β-CD was added at the molar ratio of 1:1, the content of NP-40 in NP-40/SDS compound system could be accurately detected by UV spectrometry, the recovery rate was 99.09% ~ 100.00%. The experimental results of Job"s, FT-IR and ¹H-NMR showed that NP-40 entered the cavity of HP-β-CD molecular and formed a 1:1 inclusion complex, which was the fundamental reason for HP-β-CD to eliminate the interference of SDS on the UV spectrum of NP-40 and destroy NP-40 to form micelles.