The interactions of myoglobin (Mb) and its mutant Mb(D60K) with two surfactants were studied using stopped-flowfluorescence spectroscopy, ultraviolet-visible (UV-Vis) absorption spectroscopy, fluorescence spectroscopy, and circular dichroism(CD) spectroscopy. The Mb external asparagine 60 into lysine, stopped-flow fluorescence results showed that the interactions of different concentrations of sodium dodecyl sulfate (SDS) and cetyltrimethyl ammonium bromide (CTAB) with Mb and Mb(D60K) were all quasi-first order reactions. Although Mb(D60K) was simply altered, the remarkable differences observed indicated that amino acid 60 influences the protein function greatly. Furthermore, results fromUV-Vis spectroscopy, fluorescence spectroscopy and CD spectroscopy all indicated that the configurations and functions ofMb andMb (D60K) were changed by the surfactants. The data indicates that Mb and its mutant exhibit different adaptabilities and stabilities in the surfactant solutions. From a comprehensive and comparative data analysis, we determined that the mutant D60K has more stable and adaptable functional and structural properties.