The objective of this study was to identify the correlation between protein molecular structures and protein nutritional profiles of co-products from cereal grains after bio-fuel and bio-brewing processing in terms of (1) protein chemical profiles; (2) protein subfractions; (3) total digestible protein; (4) protein degradation and estimated intestinal CP digestibility. Five kinds of corn DDGS and 2 kinds of barley DDGS were collected from different manufactures in the north of China from 2012 to 2013. The protein molecular structure spectral feature were analyzed using advanced molecular spectroscopy technique at University of Saskatchewan. The protein subfractions were partitioned using CNCPS system. Total digestible protein, protein rumen degradation and intestinal digestion were determined using dry Holstein dry cows at University of Saskatchewan. Statistical analysis was performed using the PROC MIXED of SAS 9.3. The results showed that the co-products from corn and barley grains differed in both protein nutritional profiles and protein molecular structures in terms of α-helix, β-sheet spectral intensity and their ratio and amide I, amide II spectral intensity and their ratio. Protein amide II height had a positive correlation with (P < 0.05) PB2 fraction with R = 0.53, but that other protein amide parameters had no correlation with (P > 0.05) PA, PB1, PB3 and PC fractions. Protein amide II height had a positive correlation with (P < 0.05) TDN with R = 0.74. Protein amide II height has a negative correlation with (P < 0.05) protein degradability (R_DCP) with R = −0.67, and a positive correlation with (P < 0.05) intestinal protein digestibility (I_DCP) with R = 0.60 and total-tract available protein (T_ACP) with R = 0.58. For protein secondary structure, the α-helix to β-sheet ratio was negatively correlated with (P < 0.05) total protein digestibility (T_DCP) with R = −0.56 and positively correlated with (P < 0.05) total digestible crude protein (tdCP) with R = 0.55. In conclusion, protein molecular spectral parameters in the co-products are association with protein nutritional profiles and protein degradation and digestion.