Black shale deposited in the transitional period from the Late Ordovician to Early Silurian is the most important source rock and shale gas reservoir in the Yangtze region of South China. However, the source of these sediments is still controversial. In this paper, the changes in total organic carbon (TOC), total sulfur (TS), organic carbon isotopes (δ13Corg), biomarkers, trace elements, and rare earth elements in the Ordovician–Silurian boundary strata of the XK-1 well in northern Guizhou Province, South China, have been systematically studied. The paleoenvironmental and paleoclimatic conditions of the Late Ordovician to Early Silurian and their relationship with organic matter enrichment in the Upper Yangtze Platform have been reconstructed. The distribution of biomarkers reflects that the Late Ordovician–Early Silurian shale was deposited in the marine environment and was highly contributed by marine plankton/algae and microorganisms. Paleoclimatic proxies (Sr/Cu, δ13Corg) show that the global climate system experienced significant changes from a warm-humid climate to a brief period of cold-dry climate and then back to a warm-humid climate during the Ordovician–Silurian transition. This warm and humid climate condition helps to improve the biological productivity within the photic zone of the water column during deposition. In addition, the low oxygen (reduction) conditions during the deposition of the Late Ordovician–Early Silurian deposits are characterized by low Pr/Ph values (0.39–0.79) and relatively high elemental ratios of V/Ni (1.40–5.77) and V/(V + Ni) (0.58–0.85). This paleoredox condition contributes to the preservation of organic matter during deposition of the Late Ordovician–Early Silurian deposits. Therefore, it is demonstrated that the climate and ocean fluctuated greatly during the Late Ordovician–Early Silurian transition period, and this fluctuation provided necessary control factors for marine anoxia, primary productivity, and subsequent organic-rich black shale deposition in the Upper Yangtze region during the Late Ordovician and Early Silurian intervals.