The purpose of this experiment was to evaluate changes in fermentation quality, chemical composition, aerobic stability, anti-nutritional factors, and in situ disappearance characteristics of various protein-based total mixed rations. Soybean meal (control, non-fermented), fermented cottonseed meal (F-CSM), and fermented rapeseed meal (F-RSM) group were used to prepare the TMRs with corn, whole-plant corn silage, corn stalks, wheat bran, and premix. The test groups were inoculated at 50% moisture with Bacillus clausii and Saccharomyces cariocanus and stored aerobically for 60 h. The nylon-bag method was used to measure and study the rumen's nutrient degradation. The pH of all TMRs after 48 h of air exposure was below 4.8, whereas that of the F-CSM and control and F-RSM groups increased to 5.0 and >7.0, respectively. After 8 h of aerobic exposure, the temperatures of all groups significantly increased, and 56 h later, they were 2 °C higher than the surrounding air. The lactic acid concentration in the F-CSM and F-RSM groups increased after 12 h of aerobic exposure and then decreased. The acetic acid concentrations in the fermented groups decreased significantly with the increasing air-exposure time. The yeast population of the TMRs increased to more than 8.0 log10 CFU/g before 72 h of air exposure, followed by a decrease in the population (5.0 log10 CFU/g). After fermentation, the free gossypol (FG) concentration in F-CSM decreased by half and did not change significantly during the air-exposure period. Fermentation with probiotics also reduced the F-RSM's glucosinolate concentration, resulting in a more than 50% detoxification rate. Compared with the F-CSM and F-RSM groups, the effective degradation rates of nutrients in the control group were the lowest, and the dry matter (DM), crude protein (CP), natural detergent fiber (NDF), and acid detergent fiber (ADF) all degraded effectively at rates of 28.4%, 34.5%, 27.8%, and 22.8%, respectively. Fermentation with B. clausii and S. cariocanus could improve the fermentation quality and nutrient composition, decrease the anti-nutritional factor, and increase nutrient degradation of the TMR with cottonseed meal or rapeseed meal as the main protein source, thus achieving detoxification.