beta-Alanine is an important precursor for the production of food additives, pharmaceuticals, and nitrogen-containing chemicals. Compared with the conventional chemical routes for beta-alanine production, the biocatalytic routes using l-aspartate-alpha-decarboxylase (ADC) are more attractive when energy and environment are concerned. However, ADC's poorly understood properties and its inherent mechanism-based inactivation significantly limited the application of this enzyme. In this study, three genes encoding the ADC enzymes from Escherichia coli, Corynebacterium glutamicum, and Bacillus subtilis were overexpressed in E. coli. Their properties including specific activity, thermostability, and mechanism-based inactivation were characterized. The ADC enzyme from B. subtilis, which had higher specific activity and thermostability than the others, was selected for further study. In order to improve its activity and relieve its mechanism-based inactivation by molecular engineering so as to improve its catalytic stability, a high-throughput fluorometric assay of beta-alanine was developed. From a library of 4000 mutated enzymes, two variants with 18-22% higher specific activity and 29-64% higher catalytic stability were obtained. The best variant showed 50% higher beta-alanine production than the wild type after 8 h of conversion of l-aspartate, showing great potential for industrial biocatalytic production of beta-alanine.