Soybean is an important legume crop with high protein content, but deficient in sulphur (S) containing amino acids viz methionine and cysteine. Soybean protein quality can be improved by nutrient fertilization. Present studies report the effect of nitrogen (N) and S nutrition on nodulation, activities of ammonia assimilating enzymes and biochemical parameters in soybean roots and nodules. Nitrogen as urea @ 31.25 kg ha^-1 and sulphur as gypsum @ 20 kg ha-1 alone significantly increased nodule number, fresh and dry weight per nodule, root length and nodular hemoglobin content whereas total soluble proteins, free amino acids, glucose and sucrose content did not vary significantly in nodules and roots. Treatment of N or S significantly increased glutamate dehydrogenase activity in cytosol and bacterial fractions from 70 to 90 days after sowing whereas aspartate aminotransferase, glutamate synthase, glutamine synthetase and alanine aminotransferase activities showed non-significant variations in roots at different stages of development under the influence of N or S alone or their combination. Results suggested that N and S could improve nodulation and vegetative growth in soybean, but ammonia assimilating enzyme activities in roots/nodules did not vary significantly.