Abstract: Agricultural straw has surged as one typical by-product of crops with the ever-increasing grain demand. These straws have the great potential to be one of the most important feedstocks for biogas and hydrogen production through anaerobic digestion in the future. The effective treatment of straw can also be realized to produce biofuel and composite organic fertilizer. Among them, the main components of straw are cellulose, hemicellulose, and lignin. However, the cellulose and hemicellulose in the straw are fused to form the complex polymers, whereas, the lignin can form the complex lignocellulosic carbohydrates in the outermost layers of cellulose and hemicellulose. Both cellulose and hemicellulose can be encapsulated in the inner layers under the strong interaction forces between the molecules and between hydrogen and covalent bonds within the molecular structure. As such, the energy conversion rate of straw can decrease during anaerobic digestion. Therefore, the straw must be pretreated to break the lignin structure before anaerobic digestion. Fortunately, freeze-thaw pretreatment can be expected to simply operate in this case. However, low-temperature refrigeration has high energy consumption, while the commonly used acid-alkali soaking solution is easy to cause the potential risk of environmental pollution. In this study, the soaking solution was taken as the biogas slurry rich in ammonia nitrogen, one of the by-products during anaerobic digestion. The natural average temperature (−20 ℃) was used to simulate northern China in winter. A systematic investigation was made to clarify the effects of biogas slurry soaking and freeze-thaw pretreatment on the physicochemical properties of rice straw, in terms of soaking temperature, soaking liquid-solid ratio, soaking time, and the number of freeze-thaw cycles. The results showed that there was a damaged lignin structure and hydrogen bond of lignocellulose molecules after the pretreated rice straw. A weakening trend was also found in the absorption peaks of O-H, C-H, C=C, C=N, N=N, and N=O groups. The crystal structure of rice straw was almost unchanged, but the crystallinity of rice straw decreased significantly, reaching 29.82% and decreasing by 21.38%, compared with the untreated straw. The C/N ratio of rice straw was significantly reduced to below 30 during biogas slurry immersion. By contrast, there was a relatively small effect of freeze-thaw treatment on the C/N ratio of the immersed rice straw, which was reduced to the minimal 28.19 suitable for anaerobic digestion. The removal rates of lignin, hemicellulose, and cellulose, and the volatile fatty acid (VFA) concentrations increased first and then decreased, with the increase of soaking temperature. The liquid-solid ratio showed a continuous increase trend with the increase of soaking time and freeze-thaw cycles. Soaking time and the number of freeze-thaw cycles had significant interactive effects on the lignin removal, while soaking temperature, soaking time, and liquid-solid ratio had significant interactive effects on the VFA concentrations. The maximal lignin removal rate and VFA concentration reached 40.06% and 4 140 mg/L, respectively, in the pretreatment process of biogas slurry immersion combed with freeze-thaw. Therefore, the practically feasible performance was achieved in the combined freeze-thaw pretreatment of rice straw with biogas slurry soaking. The finding can lay a theoretical foundation for the wide application of low-cost and harmless freeze-thaw pretreatment of straw in northern cold regions.