The study was conducted in vitro to investigate the effects of different ratios of Urtica cannabina and Leymus chinensis on fiber microstructure and digestibility in ruminal fluid. The experiment was divided into five groups based on the U. cannabina/L. chinensis ratios: A (0:100), B (30:70), C (50:50), D (70:30), and E (100:0). The culture medium was collected at 0, 1, 3, 6, 12, and 24 h. The results showed that: (1) in vitro crude protein degradability (IVCPD) was higher in group A, whereas in vitro neutral detergent fiber degradability (IVNDFD) was higher in group C (p < 0.05); (2) protozoa count was increased from 1 h to 3 h and decreased afterwards, with significant differences observed in several genera (p < 0.05); (3) microbial crude protein (MCP) contents at 1, 3, 6, and 24 h were higher in groups A and C (p < 0.05); (4) the basic tissue of U. cannabina was gradually degraded. At 24h, the secondary xylem vessel structure was observed in groups B and C, but not in groups D and E. In summary, there was higher neutral detergent fiber (NDF) digestibility, higher rumen MCP contents, and lower protozoa count, showing the significance of the 50:50 ratio for microbial growth and fiber digestibility.