Shear connectors ensure effective interaction between wood beams and concrete slabs of composite beams, and their properties noticeably affect the fire resistance of timber-concrete composite beams. To investigate the shear stiffness of notched connectors in glued laminated timber (GLT)-concrete composite beams under fire conditions, 16 shear tests were conducted. The effects of fire duration and notch length on shear properties of the connectors for a given spacing were studied. The fire tests indicated that the reduction of the notch length from 200 mm to 150 mm remarkably affected the failure mode of the shear specimens, changing from compression failure of notched wood to shear failure of notched concrete. The increase in fire duration reduced effective width of the notched wood, negatively affected the shear stiffness and shear capacity of the connectors, and the shear stiffness decreased more rapidly. The notch length did not have a substantial effect on the shear stiffness of connectors. Based on the experimental results, an analytical model to estimate the shear stiffness of notched connectors in GLT-concrete beam under fire conditions was established.