Acidic Golgi pH plays an important role in protein glycosylation, one of the critical quality attributes of therapeutic proteins. To determine the intracellular Golgi pH during culture, stable Chinese hamster ovary (CHO) cell clones expressing pHluorin2, a ratiometric pH‐sensitive fluorescent protein (FP), in the cis‐ and trans‐Golgi, were constructed by fusing pHluorin2 with specific targeting proteins, acetylglucosaminyltransferase, and a galactosyltransferase, respectively. Stable CHO cell clones expressing pHluorin2 in the cytoplasm were also constructed. The subcellular localization of FPs was confirmed by immunofluorescence analysis. Live‐cell imaging revealed that the intracellular pH (pHi) of clones expressing the ratiometric pH‐sensitive FPs converged to a specific pH range (cis‐Golgi: 6.4–6.5; trans‐Golgi: 5.9–6.0; and cytoplasm: 7.1–7.2). The pHi was successfully evaluated in various culture conditions. Although culture pH was maintained at 7.2 in a bioreactor, the Golgi pH increased with culture time. Elevated ammonia concentration and osmolality were partially responsible for the increased Golgi pH during bioreactor cultures. Taken together, the application of ratiometric pH‐sensitive FPs in monitoring the Golgi pH of CHO cells during culture provides a new perspective to improve protein glycosylation through pHi control.