The microbiological quality of drinking water is a concern to consumers, water suppliers, regulators, and public health authorities alike. Monitoring the microbiological quality of drinking water largely relies on the examination of indicator bacteria such as coliforms like Escherichia coli. E. coli is widely used as an indicator of fecal pollution when monitoring the microbial quality of drinking water, because it is abundant in all mammal feces and, therefore, is found in sewage and in natural waters contaminated with fecal matter, from human origin, wild animals, or derived from agricultural activity. This paper describes the development of a novel handheld fluorimeter for the rapid detection of E. coli in drinking water based on a specific cellular biomarker. The measurement system is based on a photomultiplier tube that captures the fluorescence signal produced by the cellular biomarker when it is excited by an ultraviolet LED. The cellular biomarker is also developed and it consists of a chimeric protein with a Green fluorescent protein (GFP) in the N-terminal domain and a specific amino acid sequence in the C-terminal domain (Colicin S4) that targets specifically the structure of the microorganism to be detected. The instrument is simple to use, lightweight, and can be powered by either an ac/dc power adapter or a rechargeable battery, making it an excellent choice for the rapid detection of E. coli in drinking water in field studies and laboratory measurements.