Maximum lignocellulose yield of biomass sorghum [Sorghum bicolor L. (Moench.)] is hampered by complex biological phenomena related to rotation, nitrogen (N) fertilization, soil tillage, and excessive biomass removal. The biochemical basis of the effects of agronomic practices on sorghum production was studied by the enzymology of the active peroxidase (EC 1.11.1.7) isoenzymes that synthesize lignin. All studied practices altered the peroxidase pI values. Control sorghum without rotation and without N fertilization had the most inhibited peroxidase with very low maximum velocity (Vmax) value (3.10 mmol·min^﹣1), and very low lignin (857 kg·ha^﹣1) yield, which could decrease soil organic carbon possibly leading to adverse changes in soil chemistry. Corn-sorghum rotations with and without N fertilization increased the Vmax values of peroxidase and lignin and cellulose yields. Rotated sorghum subjected to 50% residue return (the percentage of crop residue was returned to the plot immediately after grinding at harvest) and 280 kg·ha^﹣1 N fertilization possessed very active peroxidase (Vmax value 66.4 mmole·min^﹣1) and the highest lignin (1387 kg·ha^﹣1) yield. The 25% residue return rate without N fertilization induced high lignin (1125 kg·ha^﹣1) and cellulose (11,961 kg·ha^﹣1) but the 25% residue return rate with 280 kg·ha^﹣1 N fertilization induced lower lignin (1046 kg·ha^﹣1) yield. Continuously cropped sorghum treated with 336 kg·N·ha^﹣1 produced active peroxidase that shared competitive inhibition relationship with the peroxidase of the 84 kg·N·ha^﹣1 treatment. Ridge tillage combined with 280 kg·ha^﹣1 N fertilization under continuous sorghum resulted in inhibited peroxidase possessing low Vmax value (13.0 μmole·min^﹣1). Changing to conventional tillage combined with 280 kg·ha^﹣1 N fertilization relieved the inhibition and increased the Vmax value to 23.7 mmol·min^﹣1. These biological anomalies of sorghum cell wall related to agronomic practices originated from doubly inhibited sorghum peroxidases. This understanding may guide the choice of sustainable agronomic practices for maximizing lignocellulose yields for the bioenergy industry while protecting the environment.