This study was undertaken at long-term fertilizer experiment (LTFE) fields of IARI research farm, New Delhi, to assess the effect of long term fertilizer application over 35 years on the activity of selected soil enzymes, nutrient availability and acquisition during three physiological stages of wheat growth. Soil enzymes play a significant role in the transformation of nutrients and their availability to plants. Most of the researchers studied the effect of long-term fertilizer and crop management practices on soil microbial biomass and enzyme activities before or after the crop growing seasons. Since soil enzymes are much influenced by rhizospheric conditions, it is important to understand the level of soil enzyme activities and nutrient availability during various stages of plant growth. Hence this experiment was conducted to assess soil enzyme activity affecting nutrient availability and uptake in wheat due to long-term application of fertilizers and manures.