Fluorescently labelled small molecule probes (fluorescent probes) play an important role in cell imaging and are often used in combination with light-affinity probes to determine the subcellular localisation of target proteins. To investigate the target proteins of 18β-glycyrrhetinic acid (18β-GA), which regulates the macrophage inflammatory response, we designed and synthesised three types of fluorescent probes. We analysed its structure-activity relationship by evaluating the biological activity and screening for fluorescent probes with high activity. Our results showed that modifying C-3 hydroxyl and C-30 carboxyl groups enhanced the anti-inflammatory activity of 18β-GA, and found that two preferred probes had similar effects on LPS-induced, inflammation-related factor release (IL-1β, TNF-α, IL-6, HDAC8, P-STAT3, and SOCS3) to those of 18β-GA. Fluorescence signals of the preferred probes Ia and IIc were observed in the cytoplasm. The above results indicated that the anti-inflammatory site of 18β-GA may be located in proteins in the cytoplasm, which would provide useful information for research on the anti-inflammatory targets of 18β-GA.