Alfalfa silage has a high proportion of rumen-degradable protein content. Increasing dietary rumen-degradable starch (RDS) can enhance ruminal microbial protein synthesis. This study was conducted to investigate the influence of RDS levels in substrates with alfalfa silage on in vitro rumen fermentation and nitrogen (N) utilization. Rumen fluid was collected and dispensed into anaerobic fermentation bottles, each containing 1 g of substrate and 60 mL of rumen fluid–buffer mixture. The substrate was composed of 40% alfalfa silage and five different RDS levels: 14.85% RDS, 16.40% RDS, 18.67% RDS, 20.21% RDS, and 21.62% RDS. For each RDS level, three replicates were prepared. Each substrate was then incubated at 39 °C for 3, 6, 12, and 24 h. After incubation, the following parameters were measured: gas production, pH, α-amylase activity, ammonia nitrogen (NH₃-N), bacterial protein (BCP), and short-chain fatty acid (SCFA) concentrations were measured. Total gas production increased linearly with increasing RDS levels from 3 to 10 h of incubation (p < 0.01), with no difference observed among five levels after 11 h. At 3 h of incubation, pH decreased linearly with increasing RDS levels (p < 0.05). BCP concentrations and α-amylase activity increased linearly or quadratically with increasing RDS levels (p < 0.01), while the R4 group had the highest concentrations of BCP and the R5 group had the highest activity of α-amylase (p < 0.01). At 6 h of incubation, the NH₃-N concentration decreased linearly or quadratically with increasing RDS levels (p < 0.05), and the α-amylase activity, acetate, propionate, and total SCFA concentrations increased linearly (p < 0.01). The R4 group had the highest activity of α-amylase (p < 0.01), and the R5 group had the highest concentrations of acetate (p < 0.05) and propionate (p < 0.01). At 12 h of incubation, BCP, NH₃-N, and propionate concentrations, as well as α-amylase activity, increased linearly or quadratically with increasing RDS levels (p < 0.05). At 24 h of incubation, the α-amylase activity increased linearly with increasing RDS levels (p < 0.05). The highest multiple-factor associative effects index was observed in the 20.21% RDS substrate, indicating that an RDS level of 20.21% in the alfalfa silage substrate resulted in a desirable rumen N utilization.