Carbonate minerals are the most common hydrothermal alteration minerals in Mississippi Valley-type lead-zinc (MVT) deposits. They record the characteristics of ore-forming fluids and their elemental or isotopic compositions can be a vector to ore bodies. However, there are different genesis types of carbonate minerals in MVT deposits, so it is difficult to distinguish ore-related carbonate minerals from those ore-unrelated carbonate minerals only by virtue of petrographic observation. As such, additional geochemical information is helpful. This paper carried out borehole assay, petrography observation, analyses of C-O-Sr isotopes and in situ trace elements for ore-related and ore-unrelated calcite from the giant Duocaima MVT Pb-Zn deposit in Qinghai province of China. The ore-unrelated calcite that formed earlier than Pb-Zn mineralization (pre-ore, Cal1) is spatially independent of the ore zones and distributed throughout the country rocks, i.e., the Late Permian Jiushidaoban Formation limestone. It shows dark brown to brownish yellow color in cathodoluminescence (CL) images without obvious oscillatory zone. The ore-related calcite precipitated slightly younger than Pb-Zn mineralization (syn-ore, Cal2) and mainly occurs within and above the ore zones. It has granular crystal shape and the assemblage with residual vugs, with dark yellow to bright orange color in CL images with oscillatory zone. Relative to Cal2, Cal1 is more similar in C-O-Sr isotopes and elemental compositions to the wall-rock limestone, suggesting the compositions of Cal1 were greatly affected by the wall rocks during the water-rock interaction. Compared with Cal1, Cal2 has higher Mn, Fe, Zn, and Pb concentrations and U/Th and V/Cr ratios, lower Mg and Sr concentrations and LREE/HREE ratios, and weaker negative Ce anomaly. It indicates that the Cal2-related ore-forming fluid has higher temperature and relatively reduced conditions. Together with data from MVT Pb-Zn deposits worldwide, it shows that higher Mn, Fe, Zn (possibly including Pb) concentrations and lower LREE/HREE ratio can help identify ore-related carbonate minerals in MVT deposits.