Abstract: Corn stalks have been traditionally disposed of by in-situ incineration or for the production of animal feed, due to the main crop residues in northeastern China. However, the conventional treatments of corn stalks have posed a great threat to the environment and resource utilization in recent years. Alternatively, biomass materials (corn stalks) can be widely expected to replace wood preparation in paper production. Different pretreatments can be utilized to improve the quality of pulp made from corn stover. The common-used pulping pretreatment can include various physical, chemical, and biological reactions. This study aims to explore the possibility and feasibility of a co-culture procedure of anaerobic microorganisms and yeast to pretreat the corn stalks. The culture medium was firstly prepared in the experiment, and then the resulting goat rumen fluid was filtered and centrifuged, where the centrifuged supernatant was used for the identification. After that, the supernatant of rumen fluid (anaerobic fungi: Pecoramyces sp.) and Saccharomyces cerevisiae S1145 were added to the medium for the simple co-culture. The components of the naturally air-dried corn stalks were separated, where the corn stalk husks were collected, crushed, and sieved, and the shredded husks (as a substrate) were added to the medium for the simple co-cultivation at 39°C for 72 hours. The results show that the Saccharomyces cerevisiae presented the ethanol in the metabolites of fermentation broth under the action of rumen fungi, indicating that the rumen fungi and yeast were co-cultured in vitro. The content of Saccharomyces cerevisiae was greatly contributed to the metabolism. The ethanol content in the product reached the maximum when adding 5ml of Saccharomyces cerevisiae, accounting for 32.09% of the total metabolite content. The ethanol content was increased by 23.04 percentage points with Saccharomyces cerevisiae, compared with the control without. The co-culture process of anaerobic fungi and Saccharomyces cerevisiae was utilized to pretreat the corn stalk bark, where the Saccharomyces cerevisiae was improved the ethanol production. The enzyme preparation was normally used to enzymatically hydrolyze plant cellulose in the conventional second-generation cellulosic ethanol production. Nevertheless, the enzyme preparation cannot be reused during processing, leading to the high production cost of cellulosic ethanol. Taking the rumen of ruminants as a bionic object, the living environment of anaerobic microorganisms was simulated in vitro to create the conditions for the sustainable production of cellulosic ethanol. Consequently, this experiment provided a new way to treat the corn stalks. The corn stalks after biological treatment can be used to produce the biomass pulp. The experiments have verified that the rumen anaerobic microorganisms and Saccharomyces cerevisiae can self-reproduce the corn in an artificial biomimetic environment, indicating the feasibility of straw to produce ethanol. The treatment and reuse of corn stalks can greatly contribute to the economic benefits of corn stalk pulping, while reducing the overall cost consumption for the high resource utilization.