The thermal and mutual diffusion coefficients of fluid working fluids are important transfer properties that characterize their heat and mass transfer in the field of refrigeration, petroleum, chemical and others. Reliable thermal and mutual diffusion coefficient data are normally necessary for design and optimization of the equipment and process. The thermal and mutual diffusion coefficients can be determined with different equipment. The dynamic light scattering method owns the advantages of measuring properties under equilibrium condition and in the non-contact and absolute way. However, it is still not an easy way to obtain the two properties simultaneously and reliably by the dynamic light scattering method. Therefore, the present study tries to understand the influence of sampling time, incident angle, viscosity, refractive index deviation and Lewis number Le in dynamic light scattering method on the reliability and accuracy of the method. In consideration of the difference of viscosity and refractive index, three binary systems of n-Hexane/n-Decane, n-Hexane/n-Hexadecane and n-Hexane/n-Decane at the defined molar fractions (0.50/0.50, 0.06/0.94 and 0.85/0.15, respectively) are selected as reference fluids. The first and third systems have similar viscosity, but different refractive index difference of the components; the second and third systems have similar refractive index difference, but different viscosity. The measurement is performed in the saturated condition at different sampling times, incident angles and in a wide temperature range. Correlations are established based on the experimental data for both properties. The results show that the thermal diffusion coefficient and Le number decrease as the temperature increases, while the mutual diffusion coefficient increases as the temperature increases. The relaxation times of temperature fluctuations and concentration fluctuations of the binary system of n-Hexane and n-Decane with equal molar fractions satisfy a proportional relationship with the inverse of the square of the wave number, and the temperature and concentration fluctuations corresponding to the heat and mutual diffusion coefficients are verified to be consistent with the hydrodynamic model at the same time. The maximum fitting deviations between the measured thermal diffusivity and the calculated value of the fitting equation are 1.50%, 1.10% and 1.00% respectively, and the average absolute deviations are 4.00%, 0.34% and 1.95% respectively; the maximum deviations of mutual diffusion coefficient are 2.00%, 5.62% and 1.00% respectively, and the average absolute deviations are 1.06%, 2.58% and 0.31% respectively. As the sampling time is 1.5 to 3 times of the relaxation time, the incident angle is between 8° and 12°, the system has lower viscosity and higher refractive index deviation (>4%) and the Le value is 10~80, so it is possible to obtain both the thermal and mutual diffusion coefficients of the binary mixtures with an uncertainty of less than 5%. The dynamic light scattering experimental system developed in this study can be used to obtain the thermal and mutual diffusion coefficients of various complex binary systems simultaneously under the above defined conditions, providing a method for the study of diffusion in complex binary systems.