Abstract: In response to the complex and time-consuming process of existing methods for determination of sulfur in rubber, as well as the lack of certified reference materials for natural rubber matrices currently available on the market, artificial reference samples of natural rubber matrices with different sulfur content gradients were prepared using sulfur, zinc oxide, stearic acid, vulcanization accelerator N-tert-butyl-2-benzothiazole sulfonamide (TBBS), and crude rubber as raw materials through artificial batching, mixing, and vulcanization pressing. Wavelength dispersive X-ray fluorescence spectrometry was used for determination of sulfur in natural rubber to draw the calibration curve. As shown by the results, mixing for 12 min during the preparation of the reference samples helped to achieve uniform dispersion of sulfur element. The dispersion of sulfur element and the uniformity of the reference samples were the best when settling at 25 ℃ for 48 h. Linear relationship between the corresponding fluorescence intensity and mass fraction of sulfur was found in the range of 1.0%—4.0%, with detection limit of the method of 0.006% (mass fraction). This method was used to determine quality control samples 10 times in parallel, and the average value obtained was basically consistent with the theoretical value, with RSDs less than 2.0%. Compared with ion chromatography method of the national standard GB/T 41946—2022, the results of the two methods were basically consistent.