Microbiological water quality is traditionally assessed using culture-based enumeration of faecal indicator bacteria such as Escherichia coli. Despite their relative ease of use, these methods require a minimal 18-24 h-incubation step before the results are obtained. This study aimed to assess the suitability of an autonomous online fluorescence-based technology measuring β-glucuronidase (GLUC) activity for rapid near-real time monitoring of E. coli in water. The analytical precision was determined and compared to an automated microbial detection system, two culture-based assays and quantitative real-time PCR (qPCR). Using replicate measurements of grab samples containing E. coli concentrations between 50 and 2330 CFU.100 mL-1, the autonomous GLUC activity measurement technology displayed an average coefficient of variation (CV) of less than 5% that was 4-8-fold lower than other methods tested. Comparable precision was observed during online in situ monitoring of GLUC activity at a drinking water intake using three independent instruments. GLUC activity measurements were not affected by sewage or sediments at concentrations likely to be encountered during long-term monitoring. Furthermore, significant (p < 0.05) correlations were obtained between GLUC activity and the other assays including defined substrate technology (r = 0.77), membrane filtration (r = 0.73), qPCR (r = 0.55) and the automated microbial detection system (r = 0.50). This study is the first to thoroughly compare the analytical performance of rapid automated detection technologies to established culture and molecular-based methods. Results show that further research is required to correlate GLUC activity to the presence of viable E. coli as measured in terms of CFU.100 mL-1. This would allow the use of autonomous online GLUC activity measurements for rapid E. coli monitoring in water supplies used for drinking water production and recreation.