The invention relates to a two-photon material threshold testing method and device. The method comprises the steps that 1 focusing is carried out; 2 the minimum threshold m and the highest threshold qof a sample and a two-photon processing window are determined, wherein the minimum threshold m and the highest threshold q are the splitting power values of a femtosecond laser; and 3 the real powerWr of the surface of the sample and the splitting power Wp of the femtosecond laser are measured; the relationship between the real power Wr and the splitting power Wp is linearly fit into a power calibration equation v; according to the minimum threshold m, the highest threshold q and the two-photon processing window in the step 2, the power calibration equation v is used to calculate the accurate highest threshold w, the accurate minimum threshold x and the accurate two-photon processing window. The method is capable of accurately measuring the power value of the laser irradiated onto the surface of the sample to acquire the threshold of the sample.