Silkworm silk, a potential material used as the matrix of conductive fibers, has attracted extensive attention due to its excellent mechanical properties. However, researches on its thermal properties, especially in the axial direction of the single silk fibroin fiber are less. In this study, the axial thermal conductivity of a single silk fibroin fiber after coating a gold film on the surface is measured using the 3ω method. The effects of the radiation heat loss on the intrinsic thermal conductivity are eliminated by varying the length of the test samples. The results show that the thermal conductivity of the single silk fibroin fiber decreases slightly as the temperature increases from 260 to 300 K. The extracted intrinsic axial thermal conductivity is about 0.775 W/m-K at room temperature, an order of magnitude higher than most textile fibers. The method proposed in this study can be extended to accurately measure the thermal conductivity of the other non-conductive micro-fibers. •A 3ω-based technique is developed to eliminate the radiation loss and obtain the intrinsic axial thermal conductivity.•Contribution from radiation is important and should be considered for samples with large lengths and small diameters.•The kintr of the single silk fibroin fiber is found to be an order of magnitude higher than most textile fibers.