Jiuzao is the main solid by-products of Baijiu industry, which contain a high amount of underutilized cellulose and proteins. In recent years, cellulose nanofibers mixed with proteins to prepare biodegradable bio-based film materials have received widespread attention. In this study, we propose a novel method to simultaneously extract kafirin and cellulose from strong-flavor type of Jiuzao, and modify cellulose to prepare cellulose nanofibers by the TEMPO (2,2,6,6-tetramethylpiperidine-1-oxide) oxidation-pressure homogenization technique, and finally mix kafirin with cellulose nanofibers to prepare a new biodegradable bio-based composite film. Based on the analysis of one-way and response surface experiments, the highest purity of cellulose was 82.04 %. During cellulose oxidation, when NaClO was added at 25 mmol/g, cellulose nanofibers have a particle size of 80–120 nm, a crystallinity of 65.8°. Finally, kafirin and cellulose nanofibers were mixed to prepare films. The results showed that when cellulose nanofibers were added at 1 %, the film surface was smooth, the light transmittance was 60.8 %, and the tensile strength was 9.17 MPa at maximum, which was 104 % higher than pure protein film. The contact angle was 34.3°. This paper provides new ideas and theoretical basis for preparing biodegradable bio-based composite film materials, and improves the added value of Jiuzao.