Objectives To develop an efficient cost-effective screening process to improve production of glucoamylase in Aspergillus niger. Results The cultivation of A. niger was achieved with well-dispersed morphology in 48-deep-well microtiter plates, which increased the throughput of the samples compared to traditional flask cultivation. There was a close negative correlation between glucoamylase and its pH of the fermentation broth. A novel high-throughput analysis method using Methyl Orange was developed. When compared to the conventional analysis method using 4-nitrophenyl alpha-D-glucopyranoside as substrate, a correlation coefficient of 0.96 by statistical analysis was obtained. Conclusion Using this novel screening method, we acquired a strain with an activity of 2.2 x 10(3) U ml (-1), a 70% higher yield of glucoamylase than its parent strain.