In this letter, we propose a differential modulation-aided practical backscatter communication system to eliminate the overhead of backscatter channel estimation. To further enhance system performance, we leverage the index of the tag’s antenna for data mapping. Additionally, considering the non-ideal reflection coefficients (RCs) in practical tag hardware, we carefully design a novel interference cancellation (IC) strategy to remove direct link interference while enabling a two-RC tag to backscatter specific modulated symbols for the proposed differential modulation process. Experiments are conducted to verify the effectiveness of the proposed IC strategy and evaluate the performance of the proposed system.