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We prepared a series of pressed powder tablets and chalcopyrite glass standards to correct mass bias when using standard–sample bracketing for in situ sulfur isotope measurements. A femtosecond laser ablation coupled multi-collector inductively coupled plasma mass spectrometry (fsLA-MC-ICP-MS) method was used to determine the sulfur isotope composition of these standards. Chalcopyrite glass (YN411-m) was prepared by quickly quenching the fused chalcopyrite, which was melted at 1000 °C under a protective N2 atmosphere. Multiple experiments were conducted for homogeneity testing of YN411-m by fsLA-MC-ICP-MS and the external precision was 0.28‰ (n = 35). Mineral particles, pressed powder tablets, and chalcopyrite glass were used as bracketing standards when determining the δ34S of a natural chalcopyrite (GC). The results showed that the concentration, elemental composition, and crystal structure caused the matrix effect. Considering practicability, the melted glass was a more appropriate standard than the pressed powder tablet. We also found that the carrier gas flow rate, laser fluence, and spot size impacted the regularity of results. Therefore, we can obtain accurate δ34S by adjusting the laser and MC-ICP-MS parameters when using non-matrix matching standards. Furthermore, fsLA-MC-ICP-MS is advantageous for in situ sulfur isotope measurement because the sensitivity can be improved and it dramatically improves the spatial resolution (10–20 μm), which can be used to explain the origin of multi-genesis deposits by analyzing smaller mineral micro-areas, especially mineral fracture-filling sulfides later in mineralization. [ABSTRACT FROM AUTHOR]