Abstract: Abstract: Light absorption and scattering are two basic phenomena during the interaction of light with biological tissues, which are characterized by the absorption coefficient and reduced scattering coefficients. Measurement of the optical properties has always been an important research topic, because they are related to the tissue chemical constituents and physical structures. Moreover, quantification of the optical properties can help us to understand light propagation in biological tissues, interpret measurement data, optimize optical devices, and improve food quality and safety assessment. Much research on optical properties measurement has been focused on biological tissues that can be simplified as homogeneous media. However, most fruits are composed of distinct layers with different optical properties. It is thus desired that appropriate multilayer models should be used to estimate optical properties of each layer. Due to complicated analytical models and parameter estimation procedures, optical properties measurement of two-layered turbid media is prone to error, which has brought a major challenge. As a relatively new optical technique, structured illumination reflectance imaging (SIRI) has shown the capability in measuring optical properties during the last decade, due to its advantages of wide-field imaging, and depth- and resolution-varying characterization. In this research, a SIRI system, which was mainly composed of the light source, digital projector, high-performance CCD camera, liquid crystal tunable filter and sample stage, was first constructed and calibrated using two-layered samples with known optical properties. Then images of three varieties of fruit (i.e., apples, kiwifruits, and mangos) were captured using the calibrated SIRI system. Coupled with the stepwise method for image demodulation and inverse parameter estimation, optical properties of the fruit skin and flesh were estimated, respectively. Finally, the discrepancies for estimating the optical properties between the SIRI and single integrating sphere (SIS) techniques were compared, and the potential influencing factors were also analyzed and discussed. The results showed that the errors of absorption and reduced scattering coefficient estimation for the first and second layer of two-layered samples with known optical properties were less than 19% and 28%, respectively. This demonstrated that the SIRI system, coupled with the stepwise method, was able to estimate the optical properties of two-layered turbid media with an acceptable accuracy. An absorption peak was observed in the absorption coefficient spectra of the skin and flesh tissues for all three kinds of fruit, due to pigment constituents, and its peak value depended largely on the pigment content. The absorption and reduced scattering coefficients of the skin tissues were much larger than those of the flesh tissues, which was especially obvious for kiwifruits. Moreover, both the time of sliced tissue and the errors for measuring thickness had a significant effect on optical properties estimation using the SIS technique. This study provides a new method for accurate measurement of optical properties of two-layered fruits, and could lay the theoretical foundation for nondestructive detection of fruit quality (e.g., soluble solids contents and firmness) based on the tissue optical properties. Further research could focus on the measurement of two- and three-dimension optical properties maps using the depth-varying capability of the SIRI technique.