Rifaximin (RFX) is a non-absorbable antibiotic with broad-spectrum efficacy. It treats travelers’ diarrhea, irritable bowel syndrome, non-systematic bacterial diarrhea, bowel infections, overgrowth syndrome, and enteric infections. In this work, carbon dots prepared from Ziziphus spina-christi leaves’ powders are utilized as a green fluorometric biosensor for the assessment of RFX. The morphological lineaments of the prepared carbon dots were recognized by using TEM and SEM techniques. The prepared carbon dots manifest a fluorescence emission peak at 432 nm after an excitation fluorescence peak at 366 nm. The absorbance band of RFX (absorbance peaks at 370 nm and 443 nm) could be thoroughly overlapped with fluorescence excitation/emission bands of the produced carbon dots. A fluorometric tool has been designed and validated for the evaluation of RFX reliant on the inner filter effect methodology, in which the produced carbon dots act as an inner filter effect fluorophore and RFX as an inner filter effect absorber. The quenching degree in the fluorescence activity of the prepared carbon dots depended on the concentration of RFX. The analytical parameters were checked and directed for successfully applied assessment of RFX concentration in different pharmaceutical formulations. The proposed tool’s greenness and eco-friendliness profile was evaluated using the most recent greenness assessment tool, which is the complementary green analytical procedure index (Complex-GAPI) and the Analytical GREEnness metric (AGREE). Additionally, using the recently released White Analytical Chemistry (WAC) tool, the whiteness characteristic—which indicated the method’s sustainability—was investigated.