L-asparaginase has been used as anti-tumour agent for the effective treatment of acute lymphoblastic leukemia. Objective of the present work was to study the effect of process variables such as cornflour, urea, L-asparagine, glucose, di-potassium hydrogen phosphate, potassium chloride, inoculum size and pH on production of extracellular L-asparaginase by Aspergillus niger in surface fermentation. The effect of significance of these variables was studied using 2-level Plackett-Burman statistical design because traditional method of bioprocess development by studying the effect of one variable at a time is tedious, time consuming and expensive. The experimental enzyme activity was subjected to statistical analysis using MINITAB 14 software. It was found that cornflour, urea and inoculum size significantly influenced as most important factors on L-asparaginase production.