How much can we increase biomass yield by promoting land conversion from annual to perennial crops? Will increased biomass extraction for biorefineries reduce soil organic carbon (SOC) and total nitrogen (TN) stock? Which cropping system is more stable for biomass production over time? To our knowledge, no study has concurrently investigated the effects of land conversion from annual to perennial crops on biomass yield, yield stability, and changes in SOC and TN stock, which limits the understanding and application of sustainable agroecosystems producing biomass for biorefineries. Based on five-year continuous observations in central Jutland Denmark, our results showed that perennial crops significantly increased biomass yield by 19% and yield stability by 88% compared to annual crops. Perennial crops significantly increased SOC content by 4% and SOC stock by 11% at 0–100 cm depth across the five years. The opposite responses of SOC content and stock under annual and perennial crops led to even more significant differences between the crop types. Perennial crops had no effect on soil TN content and increased soil TN stock to one meter depth by 22%, whereas continuous annual crops had no effect on it. Neither annual nor perennial crops had effects on SOC and TN stock when estimated based on equivalent soil mass because the soil density increased under perennial crops. Our results showed that changes in SOC and TN stock between annual and perennial crops varied with the specific calculating methods (fixed depth/equivalent mass), thus the selected methods should be clearly defined in the future research. Increases in SOC content at one meter depth were positively correlated with biomass yield and yield stability, suggesting a win-win strategy for climate mitigation and food security. Altogether, our results highlight the potential to redesign the current cropping system for sustainable intensification by selecting proper perennial crops for green biorefineries.