The endoplasmic reticulum (ER), is an important organelle in eukaryotic cells, and is closely involved in the synthesis and processing of proteins, as well as the storage, regulation, and release of calcium. A series of signaling pathways within the ER play a crucial part in the pathogenesis of various diseases, including cancer. Thus, it is necessary to design ER-targeting probes to monitor these signaling pathways. Additionally, precision medicine also requires new ER-targeting group to facilitate the delivery of drug cargoes to the ER. However, only a limited number of ER-targeting groups have been used for the design of fluorescent probes for ER imaging in living cells, as well as the development of ER-targeted drug delivery systems. Herein, a new ER-targeting fluorescent probe (BDI-ER) was designed and prepared. BDI-ER contains the hydrophilic fluorophore, derived from the core structure of GFP, and two hydrophobic octadecane chains. The amphipathic nature of BDI-ER facilitates localization in the ER. Live cell imaging demonstrated selective localization of BDI-ER towards ER compared to other organelles. Additionally, co-localization imaging in various cell lines indicate that BDI-ER is effective at targeting the ER.