With an aim to increase the understanding about sedimentary environment and isotopic and chemical characteristics of fillings in fracture cavities with multiple compositions, we conducted scanning electron microscope (SEM), fluid inclusion testing (FIT), common and trace element chemistry, full analysis testing, isotopic compositions (δ13C, δ18O, 87Sr/86Sr), and apatite fission track testing to study the formation environment of Aksu area, Tarim Basin. According to outfield and microscope observations, combined with SEM results, three textural and compositional type fractures and cavities were distinguished. Through fine analysis of geochemistry characteristics on fractures, cavities, multiple filling periods, and environments were interpreted. Constrained by rare earth element (REE) pattern diagram, relationships between carbon and oxygen isotopes, strontium isotope, the compositional patterns, and generation environment of the fracture and cavities were determined. The results show that (1) cavity, fracture filling, and wall rock primarily consist of calcite, with a proportion of 56.85%, 80.48%, and 81.00%, respectively. (2) Four fracture sets have been distinguished in the Ordovician limestone of the karst cave, Middle-Late Caledonian (Set 1), Early Hercynian (Set 2), Indo-Yanshanian (Set 3), and Himalayan orogeny (Set 4). Two stages of cave filling deposition are distinguished. Stage I was coeval with the Middle-Late Caledonian Set 1 fractures and is attributable to the circulation of freshwater fluid. Stage II was coeval with the Early Hercynian Set 2 fractures and is attributable to deep hydrothermal fluid circulation. (3) Cavity, fracture filling, and wall rock in Ordovician strata are slightly influenced by diagenesis alteration and territorial supply. Three significant filling stages were distinguished, freshwater fluid with strong oxidizing environment (Middle-Late Caledonian), hydrothermal fluid with authigenic abnormal enrichment (indicating obvious hypoxic sedimentary water, Early Hercynian), and high-temperature hydrothermal fluid from deep earth (primarily influenced by magmatism, Indo-Yanshanian, and Himalayan).