Summary In this study, proso millet (Panicum miliaceum L., PM), which is rich in protein and has a low glycemic index, was used as the primary raw material. Our objectives were to investigate the quality changes in millet when replacing low-gluten flour in the production of biscuits and to explore the feasibility of a new type of millet biscuit. We selected four high-quality varieties of millet to make biscuits: Japonica Red millet (JR), Japonica Yellow millet (JY), Glutinous Red millet (GR), and Glutinous Yellow millet (GY). The characterization and microstructural changes in the biscuits were determined. Finally, the biscuit quality was assessed via protein network analysis, tribological characterization, and in vitro digestion. The results indicated that the GR and GY biscuits had superior protein network connectivity, with 45.12% of the protein network area. However, the porosity of the JR and JY biscuits was approximately 10% higher than that of the GR and GY biscuits; the lowest porosity was 10.33% for the GR biscuits, and the highest porosity was 11.56% for the JY biscuits. Additionally, the friction coefficients of the JR and JY biscuits were consistently higher than those of the GR and GY biscuits; the friction coefficient of the JR biscuits at 60 s reached 0.86. Moreover, the RS content was higher for the JR and JY biscuits, and the highest RS content of 51.21% was observed for the JR biscuits, whereas the RDS content was higher for the GR and GY biscuits, with the highest RDS content reaching 45.64% for the GY biscuits. This study provides a new methodological approach and a theoretical basis for investigating quality changes in biscuits.