The urgency of the study is due to the need to increase the productivity of biogas plants by intensifying the process of methane fermentation of cattle manure in mesophilic mode by adding to it the waste from biodiesel production: crude glycerine. To substantiate the rational amount of crude glycerine in the substrate, the following tasks were performed: determination of dry matter, dry organic matter, and moisture of the substrate from cattle manure with the addition of crude glycerine; conducting experimental studies on biogas yield during fermentation of cattle manure with the addition of crude glycerine with periodic loading of the substrate; and development of a biogas yield model and determination of the rational composition of crude glycerine with its gradual loading into biogas plants with cattle manure. The article presents the results of research on fermentation of substrates in a laboratory biogas plant with a useful volume of 30 L, which fermented different proportions of crude glycerine with cattle manure at a temperature of 30 °C, 35 °C, and 40 °C. The scientific novelty of the work is to determine the patterns of intensification of the process of methane fermentation of cattle manure with the addition of different portions of crude glycerine. A rapid increase in biogas yield is observed when the glycerol content is up to 0.75%. With the addition of more glycerine, the growth of biogas yield slows down. The digester of the biogas plant, where experimental studies were conducted on the fermentation of substrates based on cattle manure with the addition of co-substrates, is suitable for periodic loading of the substrate. As a rule, existing biogas plants use a gradual mode of loading the digester. Conducting experimental studies on biogas yield during fermentation of cattle manure with the addition of crude glycerine with periodic loading of the substrate makes it possible to build a mathematical model of biogas yield and determine the rational composition (up to 0.75%) of crude glycerine with its gradual loading in biogas plants. Adding 0.75% of crude glycerine to the substrate at a fermentation temperature of 30 °C allows to increase the biogas yield by 2.5 times and proportionally increase the production of heat and electricity. The practical application of this knowledge allows the design of an appropriate capacity of the biogas storage tank (gasholder).