Dynamic laser speckle technique, also termed as dynamic biospeckle, is a portable and non-invasive as well as low-cost screening tool for online or point-of-sale applications on agricultural products. A custom made dynamic laser speckle system mainly consisted of a He-Ne laser generator operating at the four wavelengths of 405 nm, 520 nm, 635 nm and 780 nm, respectively, an industrial CMOS camera for the sequential images acquisition, a computer and a height adjustable sample stage. The lighting and imaging units were placed into an illumination chamber to avoid the interference of natural light. In order to optimize the measurement conditions of dynamic laser speckle technique for the beef quality evaluation, a temporal history of speckle patterns viz . inertia moment was used as speckle activity to assess the effects of the laser incident angle, laser wavelength, and lighting intensity on beef dynamic biospeckle quality. The single factor experimental design was first applied for determining preliminary suitable lighting conditions for the later Box-Behnken experimental design. Subsequently, a response surface regression function of three lighting factors was obtained to optimize the measurement conditions for beef dynamic biospeckle. Above results demonstrated that the speckle activity could reach 476.04, the R 2 value was 0.992, root mean square error value was 8.14, bias factor ( B f ) and accuracy factor ( A f ) values were within the acceptable range of 1.0< B f