Guided by conodont biostratigraphy and unconformities observed in the field, stable carbon isotopic analysis (δ13Ccarb) was performed on 210 samples from Lower–Middle Ordovician (Tremadocian to Darriwilian) sections and wells in the Tarim Basin, NW China. The δ13C trend in the Tarim Basin sections has three distinct characteristics: (1) from the Tremadocian to the Floian, a positive shift from −1.9 ‰ to −0.2 ‰ is observed near the boundary between the Penglaiba Formation and the Yingshan Formation; (2) from the Floian to the Dapingian, a positive shift in δ13C from −3 ‰ to −0.7 ‰ occurred under large-scale sea-level rise and a change in the sedimentary environment from a restricted platform to an open platform. Changes in the conodont type are also observed in the Tabei region; and (3) from the Dapingian to the Darriwilian, δ13C first decreased and then increased, showing a negative shift at the Dapingian–Darriwilian boundary. During the Floian, δ13C decreased in the study area, while it first decreased and then increased in other regions, which may reflect local sea-level movements in response to isostatic crustal movements. Two types of positive shift were identified at the Floian–Dapingian boundary, which likely show the effects of local factors, including a disconformity, dolomitization, and platform restriction, superimposed on the global signal of the carbon isotope. Some conodont zonations and recurrent negative excursions in Tremadocian, Floian and Dapingian stages appear to be truncated by unconformities, which are accompanied by short-term subaerial exposure due to sea-level fall and local tectonic uplift.