In situ microanalysis of the sulfur (S) isotope composition and elemental distribution in sphalerite is important in geochemistry. A matrix-matched reference material is still lacking in situ microanalysis. In this study, a hydrothermal synthesis method combined with hot-press sintering processes was used to synthesize a sphalerite material Sph-LD. A large number of in situ microanalyses, including trace element concentrations and S isotope compositions, were performed on Sph-LD by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and laser ablation multi-collector inductively coupled plasma mass spectrometry (LA-MC-ICP-MS) to assess the homogeneity. The LA-MC-ICP-MS results are highly consistent and yield a mean δ³⁴S value of +17.11 ± 0.20‰ (2 SD, n = 560), which is analogous with that measured by IRMS. Microanalysis of element concentrations generally agrees with the data of ICP-MS within 10% for trace elements (relative standard deviation < 10%). These results indicate that Sph-LD is suitable to be a matrix-matched reference material available for quantitative analysis of element concentrations and S isotope measurement of sphalerite using LA-ICP-MS and LA-MC-ICP-MS. Meanwhile, the synthetic method introduces a novel concept for the development of standard materials.