In order to solve the problem of thermal conductivity test of large-size diamond, the ?100 mm sample holder of vertical mode and In-Plane mode was designed for the NETZSCH LFA467 HyperFlash instrument. The test results of the ?100 mm sample holder are proved to be credible from both principle and test results. From the theoretical analysis, it is concluded that the change of sample boundary conditions brought by the new sample holder has little effect on the test results. The thermal conductivity test results of the ?100 mm sample holder in the In-Plane mode are 6.5% higher than that of the standard sample holder, and the thermal conductivity test results of the ?100 mm sample holder in the vertical mode are 3.6% higher than that of the standard sample holder. The test results are still within the acceptable range. The ?100 mm sample holder designed in this paper can test ?100 mm samples at most, which helps to protect the integrity of samples. In this paper, the non-destructive testing of thermal conductivity of large-size diamond by laser flash method is realized.