Enzyme activity is influenced by various environmental factors which may alter the three dimensional structure of an enzyme changing its rate of activity or its ability to bind substrate. The present investigation has been carried out to study the effect of germination time, temperature, substrate and enzyme concentration, promoters and inhibitor on the urease activity in germinating seeds of Macrotyloma uniflorum. The partial purification of urease enzyme has been done by acetone fractionation method. The optimum germination time for urease activity in the present study has been found to be 4 hours and the optimum temperature 60ºC. The optimum substrate (urea) concentration of urease has been found to be at 3ml of substrate and the optimum enzyme concentration at 1ml of urease enzyme. Further urease activity has been assayed in presence of various promoters like cobalt (Co), calcium (Ca) and manganese (Mn); and also an inhibitor Copper (Cu).