A sensitive field-portable fluorimeter with incubating capability and triplicate sample chambers was designed and built. The system was optimised for the on-site analysis of E. coli in recreational waters using fluorescent based enzyme assays. The target analyte was β-d-Glucuronidase (GUS) which hydrolyses a synthetic substrate 6-Chloro-4-Methyl-Umbelliferyl-β-d-Glucuronide (6-CMUG) to release the fluorescent molecule 6-Chloro-4-Methyl-Umbelliferyl (6-CMU). The system was calibrated with 6-CMU standards. A LOD of 5nM and a resolution of less than 1nM was determined while enzyme kinetic tests showed detection of activities below 1pmolmin(-1)mL(-1) of sample. A field portable sample preparation, enzyme extraction protocol and continuous assay were applied with the system to analyse freshwater and marine samples. Results from a one day field trial are shown which demonstrated the ability of the system to deliver results on-site within a 75min period.